Compositions and methods for modification of fatty acids in soybean

ABSTRACT

Methods and compositions to modify the fatty acid profiles of seeds and the oils produced therefrom include the modification of four or more alleles of fatty acid desaturases in the genome of a plant. Compositions, polynucleotide constructs, transformed and modified host cells, and plants and seeds exhibiting altered fatty characteristics are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication Nos. 62/640,682 filed on Mar. 9, 2018, 62/721,331 filed Aug.22, 2018 and 62/753,718 filed Oct. 31, 2018, each of which isincorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“7489WOPCT_SEQLIST_2019.txt” created on Feb. 26, 2019 and having a sizeof 92 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

BACKGROUND

Soybean oil produced in the US is extracted from soybean seeds and hasits major use in food products such as cooking oils, shortenings andmargarines. The soybean oil can be refined, bleached and deodorized(RBD) and may be hydrogenated to facilitate its use in shortenings. Itis nutritionally desirable to produce soybean oils rich inmonounsaturated fatty acids with reduced linolenic acid and saturatedfatty acids. Recent advances in plant genetic engineering havefacilitated the engineering of plants to have improved seed composition,such as improved fatty acid content and composition.

SUMMARY

Provided are methods of altering the fatty acid profile in the seed of aplant, such as an oil seed crop such as soybean, by introducing four ormore nucleotide modifications through four or more targeted DNA breaksat four or more genomic loci of a plant, which loci includepolynucleotides encoding a FAD2 and a FAD3 polypeptide, such as FAD2-1A,FAD2-1B, FAD3a and FAD3b. The oleic acid content in the seed isincreased and the linolenic acid content is decreased compared to theseed of a control plant not comprising the one or more introducedgenetic modifications. The modifications can be introduced throughtargeted DNA breaks at the same time in a reaction vessel and can beintroduced using no more than two guide RNAs. In some embodiments, themodifications target more than four distinct genomic loci that areinvolved in fatty acid metabolism.

The increase in oleic acid content in the seed or oil produced therefromcan be about 70% to about 90% by weight of the total fatty acids and thedecrease in linolenic acid content can be less than about 3% by weightof the total fatty acids. In some embodiments, the yield or standardagronomic performance of the plant is not affected by the modificationsor altered fatty acid profile.

In some embodiments, the modifications are targeted such that more thanone genetic modifications are present within the same coding region;non-coding region; regulatory sequence; or untranslated region of anendogenous polynucleotide encoding a polypeptide that is involved infatty acid metabolism. In some embodiments, the target site comprisesSEQ ID NO: 6 or SEQ ID NO:7. In some embodiments, the double strandbreak is induced by using a guide RNA that corresponds to a targetsequence selected from the group consisting of SEQ ID NOS: 6 and 7.

In some embodiments, the polynucleotides encode a polypeptide comprisingan amino acid sequence that is at least 90% identical to SEQ ID NOS: 70,72 or a combination thereof, and may include at least one of SEQ ID NOS:35-58, 77, 84-118 and 136-142 and an amino acid sequence that is atleast 90% identical to SEQ ID NOS: 74, 76 or a combination thereof andmay include at least one of SEQ ID NOS: 59-64, 78, 79, 119-135 and143-145. The first polynucleotide may comprise SEQ ID NO: 54, the secondpolynucleotide may comprise SEQ ID NO: 60, the third polynucleotide maycomprise SEQ ID NO: 57, and the fourth polynucleotide may comprise SEQID NO: 63. In some embodiments, the polynucleotides comprise SEQ ID NO:55, SEQ ID NO: 61, SEQ ID NO: 58, and SEQ ID NO: 64.

In some embodiments, the genomic loci comprise an edit in apolynucleotide that encodes a FAD3 or FAD2-1 polypeptide comprising anamino acid sequence that is at least 90% identical SEQ ID NOS: 74 or 76or of SEQ ID NOS: 70 or 72 such that the edit results in reducedexpression of a polynucleotide encoding the FAD3 or FAD2-1 polypeptide,reduced activity of the FAD3 or FAD2-polypeptide, generation of one ormore alternative spliced transcripts of a polynucleotide encoding theFAD3 or FAD2-1 polypeptide, deletion of one or more active sites of theFAD3 or FAD2-1 polypeptide, frameshift mutation in one or more exons ofa polynucleotide encoding the FAD3 or FAD2-1 polypeptide, deletion of asubstantial portion of the polynucleotide encoding the FAD3 or FAD2-1polypeptide or deletion of the polynucleotide encoding the full-lengthFAD3 or FAD2-1 polypeptide, repression of an enhancer motif presentwithin a regulatory region encoding the FAD3 or FAD2-1 polypeptide, ormodification of one or more nucleotides or deletion of a regulatoryelement operably linked to the expression of the polynucleotide encodingthe FAD3 or FAD2-1 polypeptide, wherein the regulatory element ispresent within a promoter, intron, 3′UTR, terminator, or any combinationthereof.

Examples of FAD2 polynucleotides which can be modified include theseed-preferred or seed-specific sequences FAD2-1A, FAD2-1B and thesequences FAD2-2A, FAD2-2B, FAD2-2C, FAD2-2D and FAD2-2E which tend tobe constitutively expressed. Examples of FAD3 polynucleotides which canbe modified include the seed-preferred sequences FAD3A, FAD3B and thesequences FAD3C and FAD3C-x2 which tend to be constitutively expressed.

Provided are soybean plants comprising four genomic loci, each locuscomprising one or more mutations compared to a control plant andencoding a polypeptide that is at least 95% identical to the amino acidsequence of SEQ ID NOS: 70, 72, 74 and 76 and having an altered fattyacid profile and containing no heterologous or foreign DNA at themodified genomic loci. In some embodiments, the four genomic locicomprise SEQ ID NOS: 54, 57, 60, and 63, comprise SEQ ID NOS: 55, 58,61, and 64 or comprise SEQ ID NOS: 77, 57, 78 and 79. In someembodiments, the oleic acid content in a seed produced by the plant isincreased to about 70% to about 90% by weight of the total fatty acidsand/or the linolenic acid content in a seed produced by the plant isdecreased to less than about 3% by weight of the total fatty acids. Insome embodiments, the soybean plant having an altered fatty acid profiledoes not have substantially affected yield.

In some embodiments, a container is provided comprising a first guideRNA sequence that targets at two different genomic loci of a plant cell,which loci comprise a polynucleotide that encodes a polypeptidecomprising an amino acid sequence that is at least 90% identical to SEQID NOS: 70 and 72 respectively, and a second guide RNA sequence thattargets at least a further two different genomic loci, which locicomprise a polynucleotide that encodes a polypeptide comprising an aminoacid sequence that is at least 90% identical to SEQ ID NOS: 74 and 76respectively. The first and second guide RNA sequences may comprisesequences corresponding to SEQ ID NO: 6 and SEQ ID NO:7 or a fragmentthere of, respectively. Also provided are plants and plant cells, suchas soybean plants and plant cells, and seeds produced therefromcomprising the guide RNA sequences. Also provided are recombinant DNAconstructs that express or comprise the guide RNA sequences, and plantsand plant cells such as soybean plants and plant cells and seedsproduced therefrom, comprising the DNA construct which may be stablyincorporated into the genome of the plant cell.

Provided are methods of detecting the presence of a polynucleotidecomprising SEQ ID NOs: 36-43, 45-52, 54, 55, 57, 58, 60, 61, 63, 64, 77,84-91, 93-100, 102-109, 111-118 or 136-145 which is indicative of thepresence of a deletion modification in a FAD2-1 or FAD3 allele. Themethod includes step of contacting a DNA sample obtained from a soybeanplant or part thereof with a pair of DNA primer molecules. The first inthe pair comprises at least 12 contiguous nucleotides of SEQ ID NOs:36-43, 45-52, 54, 55, 57, 58, 60, 61, 63, 64, 77, 84-91, 93-100,102-109, 111-118 or 136-145 and which includes the junction sequencenucleotides at positions 18-19 for SEQ ID NOs: 43, 123, 130 and 134;positions 19-20 for SEQ ID NO: 143; positions 20-21 for SEQ ID NOs:60-61, 63-64, 116, 120-122, 127, 131, 133 and 144; positions 20-22 forSEQ ID NO: 145; positions 21-22 for SEQ ID NOs: 78-79, 124, 125, 128,129, 132 and 135; positions 22-23 for SEQ ID NOs: 36 and 117; positions23-24 for SEQ ID NOs: 55, 94 and 100; positions 24-25 for SEQ ID NOs:106, 137 and 142; positions 25-26 for SEQ ID NOs: 47 and 88; positions26-27 for SEQ ID NOs: 46, 48, 87, 91, 98, 99, 108, 112, 114 and 141;positions 27-28 for SEQ ID NOs: 37-40, 45, 50-52, 54, 57, 58, 84, 90,93, 97, 103, 105, 107, 111, 115, 136 and 138; positions 27-29 for SEQ IDNOs: 49 and 139; positions 28-29 for SEQ ID NOs: 41, 42, 85, 86, 89, 95,96, 102, 104, 109, 113 and 118; positions 29-30 for SEQ ID NO: 140; orthe reverse complement thereof. The second DNA molecule in the pair iscomplementary to at least 12 contiguous nucleotides of soybean genomicDNA in proximity to and upstream or downstream of the binding site ofthe first DNA primer molecule, conditions which facilitate a nucleicacid amplification reaction are provided and the nucleic acidamplification reaction is performed, producing a DNA amplicon moleculewhich is detected, indicating the presence of a deletion modification ina FAD2-1 or FAD3 allele. The first DNA primer molecule in the pair cancomprise no more than 0, 1, 2, 3, 4 or 5 nucleotides following the pairof junction sequence nucleotides between which the deletion or insertionwas made.

In some embodiments, a method of screening for the presence or absenceof a polynucleotide comprising one or more of SEQ ID NOs: 36-43, 45-52,54, 55, 57, 58, 60, 61, 63, 64, 77, 84-91, 93-100, 102-109, 111-118 and136-145 in multiple genomic soybean DNA samples is provided. A pluralityof genomic soybean DNA samples are contacted with a first and a secondDNA primer molecule, comprising SEQ ID NO: 10 and 11 respectively with aDNA probe comprising SEQ ID NO.12, a first and a second DNA primermolecule comprising SEQ ID NO: 13 and 11 respectively with a DNA probecomprising SEQ ID NO: 14, a first and a second DNA primer moleculecomprising SEQ ID NO: 15 and 16 respectively with a DNA probe comprisingSEQ ID NO: 17, or a first and a second DNA primer molecule comprisingSEQ ID NO: 18 and 16 respectively with a DNA probe comprising SEQ ID NO:17. Conditions which facilitate a nucleic acid amplification reactionare provided and the nucleic acid amplification reactions are performedto produce a DNA amplicon molecule indicating the presence of awild-type FAD2-1A allele when the first and second DNA primer moleculescomprise SEQ ID NOs: 10 and 11 respectively, a wild-type FAD2-1B allelewhen the first and second DNA primer molecules comprise SEQ ID NO: 13and 11 respectively, a wild-type FAD3a allele when the first and secondDNA primer molecules comprise SEQ ID NO: 15 and 16 respectively, and awild-type FAD3b allele, when the first and second DNA primer moleculescomprise SEQ ID NO: 18 and 16 respectively. The DNA amplicon moleculesare detected, and at least one of the genomic soybean DNA samples doesnot result in the production of the DNA amplicon molecule, therebyindicating the presence of one or more of SEQ ID NOs: 36-43, 45-52, 54,55, 57, 58, 60, 61, 63, 64, 77, 84-91, 93-100, 102-109, 111-118 or136-145 in that sample. The nucleic acid reaction conditions may includeSEQ ID NO: 12, when the first and second DNA primer molecules compriseSEQ ID NOs: 10 and 11 respectively, SEQ ID NO: 14, when the first andsecond DNA primer molecules comprise SEQ ID NO: 13 and 11, SEQ ID NO:17, when the first and second DNA primer molecules comprise SEQ ID NO:15 and 16 respectively, and SEQ ID NO: 17, when the first and second DNAprimer molecules comprise SEQ ID NO: 18 and 16 respectively.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

The disclosure can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing that forma part of this application, which are incorporated herein by reference.

FIG. 1A shows edits made to the FAD2-1A coding sequence as describedherein, such as in the Mega74 experiment.

FIG. 1B shows edits made to the FAD2-1B coding sequence coding sequenceas described herein, such as in the Mega74 experiment

FIG. 2 shows edits made to the FAD2-1, FAD2-1B, FAD3a and FAD3b codingsequences as described herein, such as in the Mega82 Experiment

FIG. 3A shows the edits made to the FAD2-1A coding sequence as describedherein, such as the RV019927 experiment of Example 7.

FIG. 3B shows the edits made to the FAD2-1B coding sequence as describedherein, such as the RV019927 experiment of Example 7.

FIG. 4A shows the edits made to the FAD2-1A coding sequence as describedherein, such as the RV019929 experiment of Example 7.

FIG. 4B shows the edits made to the FAD2-1B coding sequence as describedherein, such as the RV019929 experiment of Example 7.

FIG. 5A shows the edits made to the FAD3a coding sequence as describedherein, such as the RV019929 experiment of Example 7.

FIG. 5B shows the edits made to the FAD3b coding sequence as describedherein, such as the RV019929 experiment of Example 7.

The sequence descriptions summarize the Sequence Listing attachedhereto, which is hereby incorporated by reference. The Sequence Listingcontains one letter codes for nucleotide sequence characters and thesingle and three letter codes for amino acids as defined in theIUPAC-IUB standards described in Nucleic Acids Research 13:3021-3030(1985) and in the Biochemical Journal 219(2):345-373 (1984).

TABLE 1 Sequence Listing Description FAD2 and 3 nucleotide andpolypeptide sequences; other fatty acid target sequences SEQ ID NOSDescription SEQ ID NO: 1 Nucleotide sequence of soybean codon optimizedCas9 SEQ ID NO: 2 Amino acid sequence of the SV40 nuclear localizationsignal SEQ ID NO: 3 Amino acid sequence of the VirD2 nuclearlocalization signal SEQ ID NO: 4 Nucleotide sequence of soybeanconstitutive promoter GM-EF1A2 SEQ ID NO: 5 Nucleotide sequence ofsoybean GM-U6-13.1 promoter SEQ ID NO: 6 Nucleotide sequence ofGM-FAD2-1 CR1 SEQ ID NO: 7 Nucleotide sequence of GM-FAD3 CR2 SEQ ID NO:8 Nucleotide sequence of RTW1211 construct SEQ ID NO: 9 Nucleotidesequence of RTW1312 construct SEQ ID NO: 10 Nucleotide sequence ofFAD2-F1 primer SEQ ID NO: 11 Nucleotide sequence of FAD2-R1 primer SEQID NO: 12 Nucleotide sequence of FAD2-T1 probe SEQ ID NO: 13 Nucleotidesequence of FAD2-F2 primer SEQ ID NO: 14 Nucleotide sequence of FAD2-T2probe SEQ ID NO: 15 Nucleotide sequence of FAD3-F1 primer SEQ ID NO: 16Nucleotide sequence of FAD3-R2 primer SEQ ID NO: 17 Nucleotide sequenceof FAD3-T2 probe SEQ ID NO: 18 Nucleotide sequence of FAD3-F2 primer SEQID NO: 19 Nucleotide sequence of Cas9-F primer SEQ ID NO: 20 Nucleotidesequence of Cas9-R primer SEQ ID NO: 21 Nucleotide sequence of Cas9-Tprobe SEQ ID NO: 22 Nucleotide sequence of PinII-99F primer SEQ ID NO:23 Nucleotide sequence of PinII-13R primer SEQ ID NO: 24 Nucleotidesequence of PinII-69T probe SEQ ID NO: 25 Nucleotide sequence ofSIP-130F primer SEQ ID NO: 26 Nucleotide sequence of SIP-198R primer SEQID NO: 27 Nucleotide sequence of SIP-170T probe SEQ ID NO: 28 Nucleotidesequence of PCR primer WOL1007 SEQ ID NO: 29 Nucleotide sequence of PCRprimer WOL1008 SEQ ID NO: 30 Nucleotide sequence of PCR primer WOL1009SEQ ID NO: 31 Nucleotide sequence of PCR primer WOL1100 SEQ ID NO: 32Nucleotide sequence of PCR primer WOL1101 SEQ ID NO: 33 Nucleotidesequence of PCR primer WOL1102 SEQ ID NO: 34 Nucleotide sequence of PCRprimer WOL1103 SEQ ID NO: 35 Nucleotide sequence of FAD2-1A WT allelenear GM-FAD2-1 CR1 site SEQ ID NO: 36 Nucleotide sequence of FAD2-1Aedited allele of the 1.1 variant near GM-FAD2-1 CR1 site SEQ ID NO: 37Nucleotide sequence of FAD2-1A edited allele of the 1.2 variant nearGM-FAD2-1 CR1 site SEQ ID NO: 38 Nucleotide sequence of FAD2-1A editedallele of the 1.3 variant near GM-FAD2-1 CR1 site SEQ ID NO: 39Nucleotide sequence of FAD2-1A edited allele of the 6.1 variant nearGM-FAD2-1 CR1 site SEQ ID NO: 40 Nucleotide sequence of FAD2-1A editedallele of the 1.4 variant near GM-FAD2-1 CR1 site SEQ ID NO: 41Nucleotide sequence of FAD2-1A edited allele of the 1.5 variant nearGM-FAD2-1 CR1 site SEQ ID NO: 42 Nucleotide sequence of FAD2-1A editedallele of the 1.6 variant near GM-FAD2-1 CR1 site SEQ ID NO: 43Nucleotide sequence of FAD2-1A edited allele of the 1.7 variant nearGM-FAD2-1 CR1 site SEQ ID NO: 44 Nucleotide sequence of FAD2-1B WTallele near GM-FAD2-1 CR1 site SEQ ID NO: 45 Nucleotide sequence ofFAD2-1B edited allele of the 1.1 variant near GM-FAD2-1 CR1 site SEQ IDNO: 46 Nucleotide sequence of FAD2-1B edited allele of the 1.2 variantnear GM-FAD2-1 CR1 site SEQ ID NO: 47 Nucleotide sequence of FAD2-1Bedited allele of the 1.3 variant near GM-FAD2-1 CR1 site SEQ ID NO: 48Nucleotide sequence of FAD2-1B edited allele of the 6.1 variant nearGM-FAD2-1 CR1 site SEQ ID NO: 49 Nucleotide sequence of FAD2-1B editedallele of the 1.4 variant near GM-FAD2-1 CR1 site SEQ ID NO: 50Nucleotide sequence of FAD2-1B edited allele of the 1.5 variant nearGM-FAD2-1 CR1 site SEQ ID NO: 51 Nucleotide sequence of FAD2-1B editedallele of the 1.6 variant near GM-FAD2-1 CR1 site SEQ ID NO: 52Nucleotide sequence of FAD2-1B edited allele of the 1.7 variant nearGM-FAD2-1 CR1 site SEQ ID NO: 53 Nucleotide sequence of FAD2-1A WTallele near GM-FAD2-1 CR1 site SEQ ID NO: 54 Nucleotide sequence ofFAD2-1A edited allele of the 3.1 variant near GM-FAD2-1 CR1 site SEQ IDNO: 55 Nucleotide sequence of FAD2-1A edited allele of the 5.3 variantnear GM-FAD2-1 CR1 site SEQ ID NO: 56 Nucleotide sequence of FAD2-1B WTallele near GM-FAD2-1 CR1 site SEQ ID NO: 57 Nucleotide sequence ofFAD2-1B edited allele of the 3.1 and 1.5a variants near GM-FAD2-1 CR1site SEQ ID NO: 58 Nucleotide sequence of FAD2-1B edited allele of the5.3 variant near GM-FAD2-1 CR1 site SEQ ID NO: 59 Nucleotide sequence ofFAD3a WT allele near GM-FAD3 CR2 site SEQ ID NO: 60 Nucleotide sequenceof FAD3a edited allele of the 3.1 variant near GM-FAD3 CR2 site SEQ IDNO: 61 Nucleotide sequence of FAD3a edited allele of the 5.3 variantnear GM-FAD3 CR2 site SEQ ID NO: 62 Nucleotide sequence of FAD3b WTallele near GM-FAD3 CR2 site SEQ ID NO: 63 Nucleotide sequence of FAD3bedited allele of the 3.1 variant near GM-FAD3 CR2 site SEQ ID NO: 64Nucleotide sequence of FAD3b edited allele of the 5.3 variant nearGM-FAD3 CR2 site SEQ ID NO: 65 Nucleotide sequence of soybean FAD2-1Agene. SEQ ID NO: 66 Nucleotide sequence of soybean FAD2-1B gene SEQ IDNO: 67 Nucleotide sequence of soybean FAD3a gene SEQ ID NO: 68Nucleotide sequence of soybean FAD3b gene SEQ ID NO: 69 Nucleotidesequence of soybean FAD2-1A coding sequence (CDS) SEQ ID NO: 70 Aminoacid sequence of soybean FAD2-1A gene SEQ ID NO: 71 Nucleotide sequenceof soybean FAD2-1B coding sequence (CDS) SEQ ID NO: 72 Amino acidsequence of soybean FAD2-1B gene SEQ ID NO: 73 Nucleotide sequence ofsoybean FAD3a coding sequence (CDS) SEQ ID NO: 74 Amino acid sequence ofsoybean FAD3a gene SEQ ID NO: 75 Nucleotide sequence of soybean FAD3bcoding sequence (CDS) SEQ ID NO: 76 Amino acid sequence of soybean FAD3bgene SEQ ID NO: 77 Nucleotide sequence of FAD2-1A edited allele of the1.5a variant near GM-FAD2-1 CR1 site SEQ ID NO: 78 Nucleotide sequenceof FAD3a edited allele of the 1.5a variant near GM-FAD3 CR2 site SEQ IDNO: 79 Nucleotide sequence of FAD3b edited allele of the 1.5a variantnear GM-FAD3 CR2 site SEQ ID NO: 80 Nucleotide sequence of SIP-130Fprimer SEQ ID NO: 81 Nucleotide sequence of SIP-198R primer SEQ ID NO:82 Nucleotide sequence of SIP-170T probe SEQ ID NO: 83 Nucleotidesequence of FAD2-1A WT allele near GM-FAD2-1 CR1 site RV019927experiment SEQ ID NO: 84 Nucleotide sequence of FAD2-1A edited allele ofthe 1.1 variant near GM-FAD2-1 CR1 site RV019927 experiment SEQ ID NO:85 Nucleotide sequence of FAD2-1A edited allele of the 1.2 variant nearGM-FAD2-1 CR1 site RV019927 experiment SEQ ID NO: 86 Nucleotide sequenceof FAD2-1A edited allele of the 1.3 variant near GM-FAD2-1 CR1 siteRV019927 experiment SEQ ID NO: 87 Nucleotide sequence of FAD2-1A editedallele of the 1.4 variant near GM-FAD2-1 CR1 site RV019927 experimentSEQ ID NO: 88 Nucleotide sequence of FAD2-1A edited allele of the 1.5variant near GM-FAD2-1 CR1 site RV019927 experiment SEQ ID NO: 89Nucleotide sequence of FAD2-1A edited allele of the 1.6 variant nearGM-FAD2-1 CR1 site RV019927 experiment SEQ ID NO: 90 Nucleotide sequenceof FAD2-1A edited allele of the 1.7 variant near GM-FAD2-1 CR1 siteRV019927 experiment SEQ ID NO: 91 Nucleotide sequence of FAD2-1A editedallele of the 1.8 variant near GM-FAD2-1 CR1 site RV019927 experimentSEQ ID NO: 92 Nucleotide sequence of FAD2-1B WT allele near GM-FAD2-1CR1 site RV019927 experiment SEQ ID NO: 93 Nucleotide sequence ofFAD2-1B edited allele of the 1.1 variant near GM-FAD2-1 CR1 siteRV019927 experiment SEQ ID NO: 94 Nucleotide sequence of FAD2-1B editedallele of the 1.2 variant near GM-FAD2-1 CR1 site RV019927 experimentSEQ ID NO: 95 Nucleotide sequence of FAD2-1B edited allele of the 1.3variant near GM-FAD2-1 CR1 site RV019927 experiment SEQ ID NO: 96Nucleotide sequence of FAD2-1B edited allele of the 1.4 variant nearGM-FAD2-1 CR1 site RV019927 experiment SEQ ID NO: 97 Nucleotide sequenceof FAD2-1B edited allele of the 1.5 variant near GM-FAD2-1 CR1 siteRV019927 experiment SEQ ID NO: 98 Nucleotide sequence of FAD2-1B editedallele of the 1.6 variant near GM-FAD2-1 CR1 site RV019927 experimentSEQ ID NO: 99 Nucleotide sequence of FAD2-1B edited allele of the 1.7variant near GM-FAD2-1 CR1 site RV019927 experiment SEQ ID NO: 100Nucleotide sequence of FAD2-1B edited allele of the 1.8 variant nearGM-FAD2-1 CR1 site RV019927 experiment SEQ ID NO: 101 Nucleotidesequence of FAD2-1A WT allele near GM-FAD2-1 CR1 site RV019929experiment SEQ ID NO: 102 Nucleotide sequence of FAD2-1A edited alleleof the 1.1 variant near GM-FAD2-1 CR1 site RV019929 experiment SEQ IDNO: 103 Nucleotide sequence of FAD2-1A edited allele of the 1.2 variantnear GM-FAD2-1 CR1 site RV019929 experiment SEQ ID NO: 104 Nucleotidesequence of FAD2-1A edited allele of the 1.3 variant near GM-FAD2-1 CR1site RV019929 experiment SEQ ID NO: 105 Nucleotide sequence of FAD2-1Aedited allele of the 1.4 variant near GM-FAD2-1 CR1 site RV019929experiment SEQ ID NO: 106 Nucleotide sequence of FAD2-1A edited alleleof the 1.5 variant near GM-FAD2-1 CR1 site RV019929 experiment SEQ IDNO: 107 Nucleotide sequence of FAD2-1A edited allele of the 1.6 variantnear GM-FAD2-1 CR1 site RV019929 experiment SEQ ID NO: 108 Nucleotidesequence of FAD2-1A edited allele of the 1.7 variant near GM-FAD2-1 CR1site RV019929 experiment SEQ ID NO: 109 Nucleotide sequence of FAD2-1Aedited allele of the 1.8 variant near GM-FAD2-1 CR1 site RV019929experiment SEQ ID NO: 110 Nucleotide sequence of FAD2-1B WT allele nearGM-FAD2-1 CR1 site RV019929 experiment SEQ ID NO: 111 Nucleotidesequence of FAD2-1B edited allele of the 1.1 variant near GM-FAD2-1 CR1site RV019929 experiment SEQ ID NO: 112 Nucleotide sequence of FAD2-1Bedited allele of the 1.2 variant near GM-FAD2-1 CR1 site RV019929experiment SEQ ID NO: 113 Nucleotide sequence of FAD2-1B edited alleleof the 1.3 variant near GM-FAD2-1 CR1 site RV019929 experiment SEQ IDNO: 114 Nucleotide sequence of FAD2-1B edited allele of the 1.4 variantnear GM-FAD2-1 CR1 site RV019929 experiment SEQ ID NO: 115 Nucleotidesequence of FAD2-1B edited allele of the 1.5 variant near GM-FAD2-1 CR1site RV019929 experiment SEQ ID NO: 116 Nucleotide sequence of FAD2-1Bedited allele of the 1.6 variant near GM-FAD2-1 CR1 site RV019929experiment SEQ ID NO: 117 Nucleotide sequence of FAD2-1B edited alleleof the 1.7 variant near GM-FAD2-1 CR1 site RV019929 experiment SEQ IDNO: 118 Nucleotide sequence of FAD2-1B edited allele of the 1.8 variantnear GM-FAD2-1 CR1 site RV019929 experiment SEQ ID NO: 119 Nucleotidesequence of FAD3a WT allele near GM-FAD3 CR2 site RV019929 experimentSEQ ID NO: 120 Nucleotide sequence of FAD3a edited allele of the 1.1variant near GM-FAD3 CR2 site RV019929 experiment SEQ ID NO: 121Nucleotide sequence of FAD3a edited allele of the 1.2 variant nearGM-FAD3 CR2 site RV019929 experiment SEQ ID NO: 122 Nucleotide sequenceof FAD3a edited allele of the 1.3 variant near GM-FAD3 CR2 site RV019929experiment SEQ ID NO: 123 Nucleotide sequence of FAD3a edited allele ofthe 1.4 variant near GM-FAD3 CR2 site RV019929 experiment SEQ ID NO: 124Nucleotide sequence of FAD3a edited allele of the 1.5 variant nearGM-FAD3 CR2 site RV019929 experiment SEQ ID NO: 125 Nucleotide sequenceof FAD3a edited allele of the 1.6 variant near GM-FAD3 CR2 site RV019929experiment SEQ ID NO: 126 Nucleotide sequence of FAD3b WT allele nearGM-FAD3 CR2 site SEQ ID NO: 127 Nucleotide sequence of FAD3b editedallele of the 1.1 variant near GM-FAD3 CR2 site RV019929 experiment SEQID NO: 128 Nucleotide sequence of FAD3b edited allele of the 1.2 variantnear GM-FAD3 CR2 site RV019929 experiment SEQ ID NO: 129 Nucleotidesequence of FAD3b edited allele of the 1.3 variant near GM-FAD3 CR2 siteRV019929 experiment SEQ ID NO: 130 Nucleotide sequence of FAD3b editedallele of the 1.4 variant near GM-FAD3 CR2 site RV019929 experiment SEQID NO: 131 Nucleotide sequence of FAD3b edited allele of the 1.5 variantnear GM-FAD3 CR2 site RV019929 experiment SEQ ID NO: 132 Nucleotidesequence of FAD3b edited allele of the 1.6 variant near GM-FAD3 CR2 siteRV019929 experiment SEQ ID NO: 133 Nucleotide sequence of FAD3b editedallele of the 1.7 variant near GM-FAD3 CR2 site RV019929 experiment SEQID NO: 134 Nucleotide sequence of FAD3b edited allele of the 1.8 variantnear GM-FAD3 CR2 site RV019929 experiment SEQ ID NO: 135 Nucleotidesequence of FAD3b edited allele of the 1.9 variant near GM-FAD3 CR2 siteRV019929 experiment SEQ ID NO: 136 Nucleotide sequence of FAD2-1A editedallele of the 1.9 variant near GM-FAD2-1 CR1 site RV019929 experimentSEQ ID NO: 137 Nucleotide sequence of FAD2-1B edited allele of the 1.9variant near GM-FAD2-1 CR1 site RV019929 experiment SEQ ID NO: 138Nucleotide sequence of FAD2-1B edited allele of the 1.10 variant nearGM-FAD2-1 CR1 site RV019929 experiment SEQ ID NO: 139 Nucleotidesequence of FAD2-1B edited allele of the 1.11 variant near GM-FAD2-1 CR1site RV019929 experiment SEQ ID NO: 140 Nucleotide sequence of FAD2-1Bedited allele of the 1.12 variant near GM-FAD2-1 CR1 site RV019929experiment SEQ ID NO: 141 Nucleotide sequence of FAD2-1B edited alleleof the 1.13 variant near GM-FAD2-1 CR1 site RV019929 experiment SEQ IDNO: 142 Nucleotide sequence of FAD2-1B edited allele of the 1.14 variantnear GM-FAD2-1 CR1 site RV019929 experiment SEQ ID NO: 143 Nucleotidesequence of FAD3a edited allele of the 1.7 variant near GM-FAD3 CR2 siteRV019929 experiment SEQ ID NO: 144 Nucleotide sequence of FAD3a editedallele of the 1.8 variant near GM-FAD3 CR2 site RV019929 experiment SEQID NO: 145 Nucleotide sequence of FAD3a edited allele of the 1.9 variantnear GM-FAD3 CR2 site RV019929 experiment

DETAILED DESCRIPTION

Compositions and methods related to modified plants, such as soybeanplants, producing seeds high in oleic acid and low linolenic acid areprovided. Suitable plants include oil seed plants, such as canola,sunflower and soybean. Plants, such as soybean plants, that have beenmodified using genomic editing techniques to produce seeds having adesirable fatty acid profile are provided. The inventors found thatmodifying four or more targeted DNA breaks at four or more genomic lociof a plant in FAD2 and FAD3 alleles, such as both FAD2-1 alleles(FAD2-1A and FAD2-1B) and both FAD3 alleles (FAD3a and FAD3b), usinggenomic editing technology as described herein provided soybean plantsthat were robust, high-yielding and produced seeds which were both highin oleic acid and low in linolenic acid. Oil produced from the seeds hadsuperior characteristics including stability, flavor profile, and fattyacid composition.

Modified seeds, such as soybean seeds, are provided with increasedlevels of oleic acid and decreased levels of linolenic acid. Thesoybeans described herein may further contain one or more of decreasedlevels of saturated fatty acids, such as one or more of palmitic andstearic acids, and decreased levels of linoleic acid.

Oils produced from seeds described herein may contain low levels ofsaturated fatty acids which are desirable in providing a healthy diet.Fats that are solid at room temperature can be used in applications suchas the production of non-dairy margarines and spreads, and variousapplications in confections and in baking. Provided are oils andtriglycerides for solid fat applications which may contain apredominance of the very high melting, long chain fatty acid stearicacid and a balance of monounsaturated fatty acid with very littlepolyunsaturated fat. Solid fat fractions having a triacylglyceridestructure with saturated fatty acids occupying the sn-1 and sn-3positions of the triglycerides and an unsaturated fatty acid at the sn-2position are provided. This overall fatty acid composition andtriglyceride structure confers an optimal solid fat crystal structureand a maximum melting point with minimal saturated fatty acid content.

The modified plants, seeds and oil compositions disclosed herein areproduced by genomic editing techniques which facilitate the editing ofthe FAD2-1A, FAD-2-1B, FAD3a and FAD3b alleles. The sense strand or thecomplement thereof may be edited.

A “FAD2”, “FAD2-1”, “FAD2-1A” or FAD2-1B″ or a “FAD2-modified plant”,“FAD2-1-modified plant”, “FAD2-1A-modified plant” or “FAD2-1B modifiedplant” generally refers to a modified plant or mutant plant that has oneor more nucleotide changes in a genomic region that encodes apolypeptide that is at least 80%, 85%, 90%, 95% or 99% identical to oneof SEQ ID NOS: 65-66 or an allelic variant thereof. A “FAD3”, “FAD3a”,“FAD3b”, “FAD3-modified plant”, “FAD3a-modified plant” or a“FAD3b-modified plant generally refers to a modified plant or mutantplant that has one or more nucleotide changes in a genomic region thatencodes a polypeptide that is at least 80%, 85%, 90%, 95% or 99%identical to one of SEQ ID NOS: 67-68 or an allelic variant thereof. Thenucleotide changes in the genomic regions of SEQ ID NOS: 65-68 caninclude modifications that result in one or more of SEQ ID NOs: 35-64being contained within the genomic region. The seeds produced by themodified plant and the oil produced therefrom shows increased oleic acidincluding for example at least about 75% or 80% oleic acid and reducedlinolenic acid, for example, less than about 3.5%, 3%, 2.5% or 2%linolenic acid.

In some embodiments the polynucleotides disclosed herein may be isolatedpolynucleotides. An “isolated polynucleotide” generally refers to apolymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA) that issingle- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases. An isolated polynucleotide in the form ofDNA may be comprised of one or more segments of cDNA, genomic DNA orsynthetic DNA.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by a single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deoxycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

A regulatory element generally refers to a transcriptional regulatoryelement involved in regulating the transcription of a nucleic acidmolecule such as a gene or a target gene. The regulatory element is anucleic acid and may include a promoter, an enhancer, an intron, a5′-untranslated region (5′-UTR, also known as a leader sequence), or a3′-UTR or a combination thereof. A regulatory element may act in “cis”or “trans”, and generally it acts in “cis”, i.e. it activates expressionof genes located on the same nucleic acid molecule, e.g. a chromosome,where the regulatory element is located. The nucleic acid moleculeregulated by a regulatory element does not necessarily have to encode afunctional peptide or polypeptide, e.g., the regulatory element canmodulate the expression of a short interfering RNA or an anti-sense RNA.

An enhancer element is any nucleic acid molecule that increasestranscription of a nucleic acid molecule when functionally linked to apromoter regardless of its relative position. An enhancer may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter.

A repressor (also sometimes called herein silencer) is defined as anynucleic acid molecule which inhibits the transcription when functionallylinked to a promoter regardless of relative position.

Promotors which may be useful in the methods and compositions providedinclude those containing cis elements, promoters functional in a plantcell, tissue specific and tissue-preferred promotors, developmentallyregulated promoters and constitutive promoters. “Promoter” generallyrefers to a nucleic acid fragment capable of controlling transcriptionof another nucleic acid fragment. A promoter generally includes a corepromoter (also known as minimal promoter) sequence that includes aminimal regulatory region to initiate transcription, that is atranscription start site. Generally, a core promoter includes a TATA boxand a GC rich region associated with a CAAT box or a CCAAT box. Theseelements act to bind RNA polymerase II to the promoter and assist thepolymerase in locating the RNA initiation site. Some promoters may nothave a TATA box or CAAT box or a CCAAT box, but instead may contain aninitiator element for the transcription initiation site. A core promoteris a minimal sequence required to direct transcription initiation andgenerally may not include enhancers or other UTRs. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic DNA segments. It is understood by those skilledin the art that different promoters may direct the expression of a genein different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions. Corepromoters are often modified to produce artificial, chimeric, or hybridpromoters, and can further be used in combination with other regulatoryelements, such as cis-elements, 5′UTRs, enhancers, or introns, that areeither heterologous to an active core promoter or combined with its ownpartial or complete regulatory elements.

The term “cis-element” generally refers to transcriptional regulatoryelement that affects or modulates expression of an operably linkedtranscribable polynucleotide, where the transcribable polynucleotide ispresent in the same DNA sequence. A cis-element may function to bindtranscription factors, which are trans-acting polypeptides that regulatetranscription.

“Promoter functional in a plant” is a promoter capable of initiatingtranscription in plant cells whether or not its origin is from a plantcell.

“Tissue-specific promoter” and “tissue-preferred promoter” are usedinterchangeably to refer to a promoter that is expressed predominantlybut not necessarily exclusively in one tissue or organ, but that mayalso be expressed in one specific cell.

“Developmentally regulated promoter” generally refers to a promoterwhose activity is determined by developmental events.

“Constitutive promoter” generally refers to promoters active in all ormost tissues or cell types of a plant at all or most developing stages.As with other promoters classified as “constitutive” (e.g. ubiquitin),some variation in absolute levels of expression can exist amongdifferent tissues or stages. The term “constitutive promoter” or“tissue-independent” are used interchangeably herein.

Provided are sequences which are heterologous nucleotide sequences whichcan be used in the methods and compositions disclosed herein. A“heterologous nucleotide sequence” generally refers to a sequence thatis not naturally occurring with the sequence of the disclosure. Whilethis nucleotide sequence is heterologous to the sequence, it may behomologous, or native, or heterologous, or foreign, to the plant host.However, it is recognized that the instant sequences may be used withtheir native coding sequences to increase or decrease expressionresulting in a change in phenotype in the transformed seed. The terms“heterologous nucleotide sequence”, “heterologous sequence”,“heterologous nucleic acid fragment”, and “heterologous nucleic acidsequence” are used interchangeably herein.

Provided are functional fragments of the sequences disclosed herein. A“functional fragment” refers to a portion or subsequence of the sequencedescribed in the present disclosure in which, the ability to modulategene expression is retained. Fragments can be obtained via methods suchas site-directed mutagenesis and synthetic construction. As with theprovided promoter sequences described herein, the functional fragmentsoperate to promote the expression of an operably linked heterologousnucleotide sequence, forming a recombinant DNA construct (also, achimeric gene). For example, the fragment can be used in the design ofrecombinant DNA constructs to produce the desired phenotype in atransformed plant. Recombinant DNA constructs can be designed for use inco-suppression or antisense by linking a promoter fragment in theappropriate orientation relative to a heterologous nucleotide sequence.

A nucleic acid fragment that is functionally equivalent to the Targetsequences of the present disclosure is any nucleic acid fragment that iscapable of modulating the expression of a coding sequence or functionalRNA in a similar manner to the Target sequences of the presentdisclosure.

The polynucleotide sequence of the targets of the present disclosure(e.g., SEQ ID NOS: 65-68) and the coding sequences SEQ ID NO: 69, 71,73, or 75, encoding the polypeptides 70, 72, 74 or 76 respectively, maybe modified or altered to reduce their expression or the characteristicsof the protein. Examples of such modifications are one or more of thesequences listed in Table 1. As one of ordinary skill in the art willappreciate, modification or alteration can also be made withoutsubstantially affecting the gene expression function. The methods arewell known to those of skill in the art. Sequences can be modified, forexample by insertion, deletion, or replacement of template sequencesthrough any modification approach. The genomic sequences contain intronsand exons which may be targeted according to the methods disclosedherein.

SEQ ID NO: 65 (soybean FAD2-1A gene) has the start codon at position 1-3and the stop codon at position 1329-1331, exon1 is from positions 1-3,intron1 is from positions 4-170, exon2 is from positions 171-1331.

SEQ ID NO: 66 (soybean FAD2-1B gene) has the start codon at position 1-3and the stop codon at position 1322-1324, exon1 is from positions 1-3,intron1 is from positions 4-163, exon2 is from positions 164-1324.

SEQ ID NO: 67 (soybean FAD3a gene) has the start codon at position 1-3and the stop codon at position 3866-3868, exon1 is from positions 1-293,intron1 is from positions 294-460, exon2 is from positions 461-550,intron2 is from positions 551-874, exon3 is from positions 875-941,intron3 is from positions 942-1076, exon4 is from positions 1077-1169,intron4 is from positions 1170-1278, exon5 is from positions 1279-1464,intron5 is from positions 1465-1756, exon6 is from positions 1757-1837,intron6 is from positions 1838-2874, exon7 is from positions 2875-3012,intron7 is from positions 3013-3685, exon8 is from positions 3686-3868.

SEQ ID NO: 68 (soybean FAD3b gene) has the start codon at position 1-3and the stop codon at position 3894-3896, exon1 is from positions 1-305,intron1 is from positions 306-497, exon2 is from positions 498-587,intron2 is from positions 588-935, exon3 is from positions 936-1002,intron3 is from positions 1003-1144, exon4 is from positions 1145-1237,intron4 is from positions 1238-1335, exon5 is from positions 1336-1521,intron5 is from positions 1522-1636, exon6 is from positions 1637-1717,intron6 is from positions 1637-1717, exon7 is from positions 2950-3087,intron7 is from positions 3088-3713, exon8 is from positions 3714-3896.

Variant promotors can be used in the methods and compositions disclosedherein. A “variant promoter” as used herein, is the sequence of thepromoter or the sequence of a functional fragment of a promotercontaining changes in which one or more nucleotides of the originalsequence is deleted, added, and/or substituted, while substantiallymaintaining promoter function. One or more base pairs can be inserted,deleted, or substituted internally to a promoter. In the case of apromoter fragment, variant promoters can include changes affecting thetranscription of a minimal promoter to which it is operably linked.Variant promoters can be produced, for example, by standard DNAmutagenesis techniques or by chemically synthesizing the variantpromoter or a portion thereof.

In some aspects of the present disclosure, the fragments ofpolynucleotide sequences disclosed herein (such as SEQ ID 65-69, 71, 73,or 75) can comprise at least about 20 contiguous nucleotides, or atleast about 50 contiguous nucleotides, or at least about 75 contiguousnucleotides, or at least about 100 contiguous nucleotides, or at leastabout 150 contiguous nucleotides, or at least about 200 contiguousnucleotides of nucleic acid sequences or polypeptides encoded by SEQ IDNOS: 69, 71, 73, or 75. In another aspect of the present disclosure, thefragments can comprise at least about 250 contiguous nucleotides, or atleast about 300 contiguous nucleotides, or at least about 350 contiguousnucleotides, or at least about 400 contiguous nucleotides, or at leastabout 450 contiguous nucleotides, or at least about 500 contiguousnucleotides, or at least about 550 contiguous nucleotides, or at leastabout 600 contiguous nucleotides, or at least about 650 contiguousnucleotides, or at least about 700 contiguous nucleotides, or at leastabout 750 contiguous nucleotides, or at least about 800 contiguousnucleotides, or at least about 850 contiguous nucleotides, or at leastabout 900 contiguous nucleotides, or at least about 950 contiguousnucleotides, or at least about 1000 contiguous nucleotides, or at leastabout 1050 contiguous nucleotides and further may include a sequencesuch as one or more of SEQ ID NOS: 1-64.

Provided are sequences that are a full complement or a full-lengthcomplement of those disclosed herein, such as the nucleotide sequencesin Table 1. The terms “full complement” and “full-length complement” areused interchangeably herein, and refer to a complement of a givennucleotide sequence, wherein the complement and the nucleotide sequenceconsist of the same number of nucleotides and are 100% complementary.

Provided are sequences that are “substantially similar” or“corresponding substantially” to those disclosed herein which can beused in the methods and compositions described herein. The terms“substantially similar” and “corresponding substantially” as used hereinrefer to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate gene expression or produce a certain phenotype. These termsalso refer to modifications of the nucleic acid fragments of the instantdisclosure such as deletion or insertion of one or more nucleotides thatdo not substantially alter the functional properties of the resultingnucleic acid fragment relative to the initial, unmodified fragment. Itis therefore understood, as those skilled in the art will appreciate,that the disclosure encompasses more than the specific exemplarysequences.

Provided are compositions and methods that includes materials, steps,features, components, or elements that consist essentially of aparticular component. The transitional phrase “consisting essentiallyof” generally refers to a composition, method that includes materials,steps, features, components, or elements, in addition to those literallydisclosed, provided that these additional materials, steps, features,components, or elements do not materially affect the basic and novelcharacteristic(s) of the claimed subject matter, e.g., one or more ofthe claimed sequences.

Isolated promoter sequences can be comprised in the methods andcompositions, such as a recombinant DNA construct, of the presentdisclosure and can be modified to provide a range of constitutiveexpression levels of the heterologous nucleotide sequence. Thus, lessthan the entire promoter regions may be utilized and the ability todrive expression of the coding sequence retained. However, it isrecognized that expression levels of the mRNA may be decreased withdeletions of portions of the promoter sequences. Likewise, thetissue-independent, constitutive nature of expression may be changed.

Modifications of the isolated promoter sequences of the presentdisclosure can provide for a range of constitutive expression of theheterologous nucleotide sequence. Thus, they may be modified to be weakconstitutive promoters or strong constitutive promoters. Generally, by“weak promoter” is intended a promoter that drives expression of acoding sequence at a low level. By “low level” is intended levels about1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000transcripts. Conversely, a strong promoter drives expression of a codingsequence at high level, or at about 1/10 transcripts to about 1/100transcripts to about 1/1,000 transcripts. Similarly, a “moderateconstitutive” promoter is somewhat weaker than a strong constitutivepromoter like the maize ubiquitin promoter.

In addition to modulating gene expression, the expression modulatingelements disclosed herein are also useful as probes or primers innucleic acid hybridization experiments. The nucleic acid probes andprimers hybridize under stringent conditions to a target DNA sequence. A“probe” is generally referred to an isolated/synthesized nucleic acid towhich, is attached a conventional detectable label or reporter molecule,such as for example, a radioactive isotope, ligand, chemiluminescentagent, bioluminescent molecule, fluorescent label or dye, or enzyme.Such detectable labels may be covalently linked or otherwise physicallyassociated with the probe. “Primers” generally referred toisolated/synthesized nucleic acids that hybridize to a complementarytarget DNA strand which is then extended along the target DNA strand bya polymerase, e.g., a DNA polymerase. Primer pairs often used foramplification of a target nucleic acid sequence, e.g., by the polymerasechain reaction (PCR) or other conventional nucleic-acid amplificationmethods. Primers are also used for a variety of sequencing reactions,sequence captures, and other sequence-based amplification methodologies.Primers are generally about 15, 20, 25 nucleotides or more, and probescan also be longer about 30, 40, 50 and up to a few hundred base pairs.Such probes and primers are used in hybridization reactions to targetDNA or RNA sequences under high stringency hybridization conditions orunder lower stringency conditions, depending on the need.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this disclosure are also definedby their ability to hybridize, under moderately stringent conditions(for example, 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplifiedherein, or to any portion of the nucleotide sequences reported hereinand which are functionally equivalent to the promoter of the disclosure.Estimates of such homology are provided by either DNA-DNA or DNA-RNAhybridization under conditions of stringency as is well understood bythose skilled in the art (Hames and Higgins, Eds.; In Nucleic AcidHybridisation; IRL Press: Oxford, U. K., 1985). Stringency conditionscan be adjusted to screen for moderately similar fragments, such ashomologous sequences from distantly related organisms, to highly similarfragments, such as genes that duplicate functional enzymes from closelyrelated organisms. Post-hybridization washes partially determinestringency conditions. One set of conditions uses a series of washesstarting with 6×SSC, 0.5% SDS at room temperature for 15 min, thenrepeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeatedtwice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. Another set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another set ofhighly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDSat 65° C.

In some embodiments, substantially similar nucleic acid sequencesencompassed by this disclosure are those sequences that are 80%identical to the nucleic acid fragments reported herein or which are 80%identical to any portion of the nucleotide sequences reported herein.Nucleic acid fragments which are at least 90% or at least 95% identicalto the nucleic acid sequences reported herein, or which are at least 90%or at least 95% identical to any portion of the nucleotide sequencesreported herein are also provided. It is well understood by one skilledin the art that many levels of sequence identity are useful inidentifying related polynucleotide sequences. Useful examples of percentidentities are those listed above, or also preferred is any integerpercentage from 71% to 100%, such as at least, at least about or about71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% and 100%.

In one embodiment, the sequences or isolated sequences of the presentdisclosure comprise a nucleotide or polypeptide sequence having at least71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% and 100% sequence identity, based on the Clustal V method ofalignment with pairwise alignment default parameters (KTUPLE=2, GAPPENALTY=5, WINDOW=4 and DIAGONALS SAVED=4), when compared to thenucleotide sequence of SEQ ID NOS: 65-69, 71, 73 or 75. It is known toone of skilled in the art that a 5′ UTR region can be altered (deletionor substitutions of bases) or replaced by an alternative 5′UTR whilemaintaining promoter activity.

Provided are substantially similar sequences useful in compositions andmethods provided herein. A “substantially similar sequence” generallyrefers to variants of the disclosed sequences such as those that resultfrom site-directed mutagenesis, as well as synthetically derivedsequences. A substantially similar promoter sequence of the presentdisclosure also generally refers to those fragments of a particularpromoter nucleotide sequence disclosed herein that operate to promotethe constitutive expression of an operably linked heterologous nucleicacid fragment. These promoter fragments comprise at least about 20contiguous nucleotides, at least about 50 contiguous nucleotides, atleast about 75 contiguous nucleotides, preferably at least about 100contiguous nucleotides of the particular promoter nucleotide sequencedisclosed herein or a sequence that is at least 95 to about 99%identical to such contiguous sequences. The nucleotides of suchfragments will usually include the TATA recognition sequence (or CAATbox or a CCAAT) of the particular promoter sequence. Such fragments maybe obtained by use of restriction enzymes to cleave the naturallyoccurring promoter nucleotide sequences disclosed herein; bysynthesizing a nucleotide sequence from the naturally occurring promoterDNA sequence; or may be obtained through the use of PCR technology.Variants of these promoter fragments, such as those resulting fromsite-directed mutagenesis, are encompassed by the compositions of thepresent disclosure.

Provided are sequences which contain one or more degenerate codons tothose provided in the sequence listing. “Codon degeneracy” generallyrefers to divergence in the genetic code permitting variation of thenucleotide sequence without affecting the amino acid sequence of anencoded polypeptide. Accordingly, the instant disclosure relates to anynucleic acid fragment comprising a nucleotide sequence that encodes allor a substantial portion of the amino acid sequences set forth herein.The skilled artisan is well aware of the “codon-bias” exhibited by aspecific host cell in usage of nucleotide codons to specify a givenamino acid. Therefore, when synthesizing a nucleic acid fragment forimproved expression in a host cell, it is desirable to design thenucleic acid fragment such that its frequency of codon usage approachesthe frequency of preferred codon usage of the host cell.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect similar oridentical sequences including, but not limited to, the Megalign® programof the LASERGENE® bioinformatics computing suite (DNASTAR® Inc.,Madison, Wis.). Unless stated otherwise, multiple alignment of thesequences provided herein were performed using the Clustal V method ofalignment (Higgins and Sharp (1989) CAB/OS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments and calculation of percent identity of proteinsequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters areKTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignmentof the sequences, using the Clustal V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

Alternatively, the Clustal W method of alignment may be used. TheClustal W method of alignment (described by Higgins and Sharp, CABIOS.5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191(1992)) can be found in the MegAlign™ v6.1 program of the LASERGENE®bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Defaultparameters for multiple alignment correspond to GAP PENALTY=10, GAPLENGTH PENALTY=0.2, Delay Divergent Sequences=30%, DNA TransitionWeight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.For pairwise alignments the default parameters areAlignment=Slow-Accurate, Gap Penalty=10.0, Gap Length=0.10, ProteinWeight Matrix=Gonnet 250 and DNA Weight Matrix=IUB. After alignment ofthe sequences using the Clustal W program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table in the same program.

In one embodiment the % sequence identity is determined over the entirelength of the molecule (nucleotide or amino acid). A “substantialportion” of an amino acid or nucleotide sequence comprises enough of theamino acid sequence of a polypeptide or the nucleotide sequence of agene to afford putative identification of that polypeptide or gene,either by manual evaluation of the sequence by one skilled in the art,or by computer-automated sequence comparison and identification usingalgorithms such as BLAST (Altschul, S. F. et al., J. Mol. Biol.215:403-410 (1993)) and Gapped Blast (Altschul, S. F. et al., NucleicAcids Res. 25:3389-3402 (1997)). BLASTN generally refers to a BLASTprogram that compares a nucleotide query sequence against a nucleotidesequence database.

The present disclosure provides genes, mutated genes, chimeric genes andrecombinant expression constructs. “Gene” includes a nucleic acidfragment that expresses a functional molecule such as, but not limitedto, a specific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” generally refers to a gene as found in naturewith its own regulatory sequences.

A “mutated gene” is a gene that has been altered through humanintervention. Such a “mutated gene” has a sequence that differs from thesequence of the corresponding non-mutated gene by at least onenucleotide addition, deletion, or substitution. In certain embodimentsof the disclosure, the mutated gene comprises an alteration that resultsfrom a guide polynucleotide/Cas endonuclease system as disclosed herein.A mutated plant is a plant comprising a mutated gene.

“Chimeric gene” or “recombinant expression construct”, which are usedinterchangeably, includes any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources.

“Coding sequence” generally refers to a polynucleotide sequence whichcodes for a specific amino acid sequence. “Regulatory sequences” referto nucleotide sequences located upstream (5′ non-coding sequences),within, or downstream (3′ non-coding sequences) of a coding sequence,and which influence the transcription, RNA processing or stability, ortranslation of the associated coding sequence. Regulatory sequences mayinclude, but are not limited to, promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

An “intron” is an intervening sequence in a gene that is transcribedinto RNA but is then excised in the process of generating the maturemRNA. The term is also used for the excised RNA sequences. An “exon” isa portion of the sequence of a gene that is transcribed and is found inthe mature messenger RNA derived from the gene, but is not necessarily apart of the sequence that encodes the final gene product.

The 5′ untranslated region (5′UTR) (also known as a translational leadersequence or leader RNA) is the region of an mRNA that is directlyupstream from the initiation codon. This region is involved in theregulation of translation of a transcript by differing mechanisms inviruses, prokaryotes and eukaryotes.

The “3′ non-coding sequences” refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

“RNA transcript” generally refers to a product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When an RNAtranscript is a perfect complimentary copy of a DNA sequence, it isreferred to as a primary transcript or it may be a RNA sequence derivedfrom posttranscriptional processing of a primary transcript and isreferred to as a mature RNA. “Messenger RNA” (“mRNA”) generally refersto RNA that is without introns and that can be translated into proteinby the cell. “cDNA” generally refers to a DNA that is complementary toand synthesized from an mRNA template using the enzyme reversetranscriptase. The cDNA can be single-stranded or converted into thedouble-stranded by using the Klenow fragment of DNA polymerase I.“Sense” RNA generally refers to RNA transcript that includes mRNA and socan be translated into protein within a cell or in vitro. “AntisenseRNA” generally refers to a RNA transcript that is complementary to allor part of a target primary transcript or mRNA and that blocksexpression or transcripts accumulation of a target gene. Thecomplementarity of an antisense RNA may be with any part of the specificgene transcript, i.e. at the 5′ non-coding sequence, 3′ non-codingsequence, introns, or the coding sequence. “Functional RNA” generallyrefers to antisense RNA, ribozyme RNA, or other RNA that may not betranslated but yet has an effect on cellular processes.

The term “operably linked” or “functionally linked” generally refers tothe association of nucleic acid sequences on a single nucleic acidfragment so that the function of one is affected by the other. Forexample, a promoter is operably linked with a coding sequence when it iscapable of affecting the expression of that coding sequence (i.e., thatthe coding sequence is under the transcriptional control of thepromoter). Coding sequences can be operably linked to regulatorysequences in sense or antisense orientation.

The terms “initiate transcription”, “initiate expression”, “drivetranscription”, and “drive expression” are used interchangeably hereinand all refer to the primary function of a promoter. As detailedthroughout this disclosure, a promoter is a non-coding genomic DNAsequence, usually upstream (5′) to the relevant coding sequence, and itsprimary function is to act as a binding site for RNA polymerase andinitiate transcription by the RNA polymerase. Additionally, there is“expression” of RNA, including functional RNA, or the expression ofpolypeptide for operably linked encoding nucleotide sequences, as thetranscribed RNA ultimately is translated into the correspondingpolypeptide.

The term “expression”, as used herein, generally refers to theproduction of a functional end-product e.g., an mRNA or a protein(precursor or mature).

The term “expression cassette” as used herein, generally refers to adiscrete nucleic acid fragment into which a nucleic acid sequence orfragment can be cloned or synthesized through molecular biologytechniques.

Expression or overexpression of a gene involves transcription of thegene and translation of the mRNA into a precursor or mature protein.“Antisense inhibition” generally refers to the production of antisenseRNA transcripts capable of suppressing the expression of the targetprotein. “Overexpression” generally refers to the production of a geneproduct in transgenic organisms that exceeds levels of production innormal or non-transformed organisms. “Co-suppression” generally refersto the production of sense RNA transcripts capable of suppressing theexpression or transcript accumulation of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020). Themechanism of co-suppression may be at the DNA level (such as DNAmethylation), at the transcriptional level, or at post-transcriptionallevel.

As stated herein, “suppression” includes a reduction of the level ofenzyme activity or protein functionality (e.g., a phenotype associatedwith a protein) detectable in a transgenic plant when compared to thelevel of enzyme activity or protein functionality detectable in anon-transgenic or wild type plant with the native enzyme or protein. Thelevel of enzyme activity in a plant with the native enzyme is referredto herein as “wild type” activity. The level of protein functionality ina plant with the native protein is referred to herein as “wild type”functionality. The term “suppression” includes lower, reduce, decline,decrease, inhibit, eliminate and prevent. This reduction may be due to adecrease in translation of the native mRNA into an active enzyme orfunctional protein. It may also be due to the transcription of thenative DNA into decreased amounts of mRNA and/or to rapid degradation ofthe native mRNA. The term “native enzyme” generally refers to an enzymethat is produced naturally in a non-transgenic or wild type cell. Theterms “non-transgenic” and “wild type” are used interchangeably herein.

“Altering expression” or “modulating expression” generally refers to theproduction of gene product(s) in plants in amounts or proportions thatdiffer significantly from the amount of the gene product(s) produced bythe corresponding wild-type plants (i.e., expression is increased ordecreased).

“Transformation” as used herein generally refers to both stabletransformation and transient transformation.

“Stable transformation” generally refers to the introduction of anucleic acid fragment into a genome of a host organism resulting ingenetically stable inheritance. Once stably transformed, the nucleicacid fragment is stably integrated in the genome of the host organismand any subsequent generation. Host organisms containing the transformednucleic acid fragments are referred to as “transgenic” organisms.“Transient transformation” generally refers to the introduction of anucleic acid fragment into the nucleus, or DNA-containing organelle, ofa host organism resulting in gene expression without genetically stableinheritance.

The term “introduced” means providing a nucleic acid (e.g., expressionconstruct) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct/expression construct) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell.

“Genetic modification” generally refers to modification of any nucleicacid sequence or genetic element by insertion, deletion, or substitutionof one or more nucleotides in an endogenous nucleotide sequence bygenome editing or by insertion of a recombinant nucleic acid, e.g., aspart of a vector or construct in any region of the plant genomic DNA byroutine transformation techniques. Examples of modification of geneticcomponents include, but are not limited to, promoter regions, 5′untranslated leaders, introns, genes, 3′ untranslated regions, and otherregulatory sequences or sequences that affect transcription ortranslation of one or more nucleic acid sequences.

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores.

Provided are plants which are dicots. The terms “dicot” and“dicotyledonous plant” are used interchangeably herein. A dicot of thecurrent disclosure includes the following families: Brassicaceae,Leguminosae, and Solanaceae.

Progeny plants are provided. “Progeny” comprises any subsequentgeneration of a plant, and can include F1 progeny, F2 progeny F3 progenyand so on.

The heterologous polynucleotide can be stably integrated within thegenome such that the polynucleotide is passed on to successivegenerations. The heterologous polynucleotide may be integrated into thegenome alone or as part of a recombinant DNA construct. The alterationsof the genome (chromosomal or extra-chromosomal) by conventional plantbreeding methods, by genome editing procedures that do not result in aninsertion of a foreign polynucleotide, or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation are also methods of modifying a host genome.

“Transient expression” generally refers to the temporary expression ofoften reporter genes such as β-glucuronidase (GUS), fluorescent proteingenes ZS-GREEN1, ZS-YELLOW1 N1, AM-CYAN1, DS-RED in selected certaincell types of the host organism in which the transgenic gene isintroduced temporally by a transformation method. The transformedmaterials of the host organism are subsequently discarded after thetransient gene expression assay.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.et al., In Molecular Cloning: A Laboratory Manual; 2^(nd) ed.; ColdSpring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989(hereinafter “Sambrook et al., 1989”) or Ausubel, F. M., Brent, R.,Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl,K., Eds.; In Current Protocols in Molecular Biology; John Wiley andSons: New York, 1990 (hereinafter “Ausubel et al., 1990”).

“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis oflarge quantities of specific DNA segments, consisting of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps comprises a cycle.

Provided are plasmids, vectors and cassettes which contain one or moreof the sequences provided, including any combination of sequencecomponents disclosed in the Examples. The terms “plasmid”, “vector” and“cassette” refer to an extra chromosomal element often carrying genesthat are not part of the central metabolism of the cell, and usually inthe form of circular double-stranded DNA fragments. Such elements may beautonomously replicating sequences, genome integrating sequences, phageor nucleotide sequences, linear or circular, of a single- ordouble-stranded DNA or RNA, derived from any source, in which a numberof nucleotide sequences have been joined or recombined into a uniqueconstruction which is capable of introducing a promoter fragment and DNAsequence for a selected gene product along with appropriate 3′untranslated sequence into a cell.

Provided are recombinant DNA constructs or recombinant expressionconstructs which contain the sequences disclosed herein, including anycombination of sequence components disclosed in the Examples. The term“recombinant DNA construct” or “recombinant expression construct” isused interchangeably and generally refers to a discrete polynucleotideinto which a nucleic acid sequence or fragment can be moved. Preferably,it is a plasmid vector or a fragment thereof comprising the promoters ofthe present disclosure. The choice of plasmid vector is dependent uponthe method that will be used to transform host plants. The skilledartisan is well aware of the genetic elements that must be present onthe plasmid vector in order to successfully transform, select andpropagate host cells containing the chimeric gene. The skilled artisanwill also recognize that different independent transformation eventswill result in different levels and patterns of expression (Jones etal., EMBO J. 4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics218:78-86 (1989)), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by PCR and Southern analysisof DNA, RT-PCR and Northern analysis of mRNA expression, Westernanalysis of protein expression, or phenotypic analysis.

Various changes in phenotype are of interest including, but not limitedto, modifying the fatty acid composition in a plant, seed or oilextracted therefrom, altering the fatty acid profile of a plant seed,altering the amounts of fatty acids in a plant seed on seed oil, and thelike as disclosed herein. Plants having a desirable phenotype and seedand oil compositions having a fatty acid profile as disclosed herein canbe generated by modulating the suppression of FAD2 and FAD3, for exampleby modulating the suppression of multiple FAD2 and FAD3 alleles, such asone, two, three or all of FAD2-1A, FAD2-1B, FAD-3a and FAD3b alleles.Target sites within the FAD2 and FAD3 alleles can be used to generateshort deletions and modifications such as described in Table 1. In anembodiment, the plants and seeds modified as disclosed herein, containonly modified genomic sequence, with no heterologous or foreign DNAremaining in the plant from the modification or in the genomic region ofthe modification or at the target site. Examples of target sites insoybean include GM-FAD2-1 CR1 (SEQ ID NO: 6) at Gm10:50014185..50014166and Gm20:35317773..35317754 and GM-FAD3 CR2 (SEQ ID NO: 7) atGm14:45939600..445939618 and Gm02:41423563..41423581.

Provided are seeds, such as soybean seeds, which can be processed toproduce oils, and the oils produced therefrom, which contain anycombination of oleic acid, linolenic acid, linoleic acid, erucic acid(C:22:1) and saturated fatty acids such as stearic acid and palmiticacid in the amounts disclosed herein. Other saturated fatty acids in thesoybean seeds and oils which may be increased or decreased compared witha control plant, seed or oil include myristic acid (C:14:0), and longchain saturated fatty acids arachidic acid (C20:0), behenic acid (C22:0)and lignoceric acid (C24:0).

Provided are seeds, such as soybean seeds, which can be processed toproduce oils, and the oils produced therefrom, which have at least or atleast about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89 or 90 percent oleic (C 18:1) acid of thetotal fatty acids by weight and less than or less than about 100, 99,98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81,80, 79, 78, 76, 75, 74, 73, 72, 71 or 70 percent oleic acid of the totalfatty acids by weight.

Provided are seeds, such as soybean seeds, which can be processed toproduce oils, and the oils produced therefrom, which have at least or atleast about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, or 3.0 percent linolenic (C 18:3) acid of the total fattyacids by weight and less than or less than about 6, 5.5, 5, 4.5, 4, 3.9,3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5,2.4, 2.3, 2.2, 2.1 or 2.0 percent linolenic acid of the total fattyacids by weight.

Provided are soybean seeds which can be processed to produce oils, andthe oils produced therefrom, which have at least or at least about 0.5,1, 2, 3, 4, 5, 6, or 7 percent linoleic (C 18:2) acid of the total fattyacids by weight and less than or less than about 55, 50, 45, 40, 35, 30,25, 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 percent linoleic acid of thetotal fatty acids by weight.

Provided are seeds, such as soybean seeds, which can be processed toproduce oils, and the oils produced therefrom, which have at least or atleast about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, or 3.0 percent stearic acid (C 18:0) of the total fattyacids by weight and less than or less than about 6, 5.5, 5, 4.5, 4, 3.9,3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5,2.4, 2.3, 2.2, 2.1 or 2.0 percent stearic acid of the total fatty acidsby weight.

Provided are seeds, such as soybean seeds, which can be processed toproduce oils, and the oils produced therefrom, which have at least or atleast about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,6.0, 6.5 or 7.0 percent palmitic acid (C 16:0) of the total fatty acidsby weight and less than or less than about 12, 11.5, 11.0, 10.5, 10.0,9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0,2.5, 2.0, 1.5, or 1.0 percent palmitic acid of the total fatty acids byweight.

Provided are seeds, such as soybean seeds, which can be processed toproduce oils, and the oils produced therefrom, which have at least or atleast about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, or 11.0 percenttotal saturated fatty acids of the total fatty acids by weight and lessthan or less than about 16, 15.5, 15, 14.5, 14, 13.5, 13.0, 12.5, 12.0,11.5, 11.0, 10.5, 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5,5.0, 4.5, 4.0, 3.5, 3.0, 2.5 or 2.0 percent total saturated fatty acidsof the total fatty acids by weight.

In an embodiment the seeds, such as soybean seeds, which can beprocessed to produce oils, and the oils produced therefrom, containelevated oleic and reduced linolenic acid as described herein, andoptionally other modified amounts of other fatty acids as describedherein.

In an embodiment, this disclosure concerns host cells comprising eitherthe recombinant DNA constructs of the disclosure as described herein orisolated polynucleotides of the disclosure as described herein. Examplesof host cells which can be used to practice the disclosure include, butare not limited to, yeast, bacteria, and plants.

Plasmid vectors comprising the instant recombinant DNA construct can beconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host cells. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene.

I. Gene Editing

In some embodiments, gene editing may be facilitated through theinduction of a double-stranded break (DSB) or single-strand break, in adefined position in the genome near the desired alteration. DSBs can beinduced using any DSB-inducing agent available, including, but notlimited to, TALENs (transcription activator-like effector nucleases),meganucleases, zinc finger nucleases, Cas9-gRNA and RNA-guided Casendonuclease systems (based on bacterial CRISPR-Cas systems, such as butnot limited to Type I-E, Cas9, Cpf1, and others), guided cpf1endonuclease systems, and the like. The DSB may be repaired via aNon-Homologous End Joining (NHEJ) pathway in the absence of anyadditional composition, via template-directed repair in the presence ofa polynucleotide modification template, or via homologous recombinationwith a heterologous polynucleotide (donor DNA molecule). The HDR pathwayrepairs double-stranded DNA breaks and includes homologous recombination(HR) and single-strand annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem.79:181-211). In some embodiments, the introduction of a DSB can becombined with the introduction of a polynucleotide modification templateor a donor DNA molecule. In some embodiments, the methods do not useTALENs enzymes or technology and plants and seeds are produced frommethods which do not use TALENs enzymes or technology.

A polynucleotide modification template can be introduced into a cell byany method known in the art, such as, but not limited to, transientintroduction methods, transfection, electroporation, microinjection,particle mediated delivery, topical application, whiskers mediateddelivery, delivery via cell-penetrating peptides, or mesoporous silicananoparticle (MSN)-mediated direct delivery.

The polynucleotide modification template can be introduced into a cellas a single stranded polynucleotide molecule, a double strandedpolynucleotide molecule, or as part of a circular DNA (vector DNA). Thepolynucleotide modification template can also be tethered to the guideRNA and/or the Cas endonuclease. Tethered DNAs can allow forco-localizing target and template DNA, useful in genome editing andtargeted genome regulation, and can also be useful in targetingpost-mitotic cells where function of endogenous HR machinery is expectedto be highly diminished (Mali et al. 2013 Nature Methods Vol. 10:957-963.) The polynucleotide modification template may be presenttransiently in the cell or it can be introduced via a viral replicon.

A “modified nucleotide” or “edited nucleotide” refers to a nucleotidesequence of interest that comprises at least one alteration whencompared to its non-modified nucleotide sequence. Such “alterations”include, for example: (i) replacement of at least one nucleotide, (ii) adeletion of at least one nucleotide, (iii) an insertion of at least onenucleotide, (iv) chemical alteration of at least one nucleotide, or (v)any combination of (i)-(iv).

The term “polynucleotide modification template” includes apolynucleotide that comprises at least one nucleotide modification whencompared to the nucleotide sequence to be edited.

A nucleotide modification can be at least one nucleotide substitution,addition or deletion. Optionally, the polynucleotide modificationtemplate can further comprise homologous nucleotide sequences flankingthe at least one nucleotide modification, wherein the flankinghomologous nucleotide sequences provide sufficient homology to thedesired nucleotide sequence to be edited.

The process for editing a genomic sequence combining DSB with or withouta modification template or using a DSB-inducing agent, such as anRNA-guided Cas endonuclease, generally comprises: providing to a hostcell a DSB-inducing agent, or a nucleic acid encoding a DSB-inducingagent, that recognizes and binds to a target sequence in the chromosomalsequence and is able to induce a DSB in the genomic sequence. In someaspects, at least one polynucleotide modification template comprising atleast one nucleotide alteration when compared to the nucleotide sequenceto be edited is provided. The polynucleotide modification template canfurther comprise nucleotide sequences flanking the at least onenucleotide alteration, in which the flanking sequences are substantiallyhomologous to the chromosomal region flanking the DSB.

In addition to the double-strand break inducing agents, site-specificbase conversions can also be achieved to engineer one or more nucleotidechanges to create one or more edits described herein into the genome.These include for example, a site-specific base edit mediated by an C⋅Gto T⋅A or an A⋅T to G⋅C base editing deaminase enzymes (Gaudelli et al.,Programmable base editing of A⋅T to G⋅C in genomic DNA without DNAcleavage. Nature (2017); Nishida et al. “Targeted nucleotide editingusing hybrid prokaryotic and vertebrate adaptive immune systems.”Science 353 (6305) (2016); Komor et al. “Programmable editing of atarget base in genomic DNA without double-stranded DNA cleavage.” Nature533 (7603) (2016):420-4. Catalytically dead dCas9 fused to a cytidinedeaminase or an adenine deaminase protein becomes a specific base editorthat can alter DNA bases without inducing a DNA break. Base editorsconvert C->T (or G->A on the opposite strand) or an adenine base editorthat would convert adenine to inosine, resulting in an A->G changewithin an editing window specified by the gRNA.

The endonuclease can be provided to a cell by any method known in theart, for example, but not limited to transient introduction methods,transfection, microinjection, and/or topical application or indirectlyvia recombination constructs. The endonuclease can be provided as aprotein or as a guided polynucleotide complex directly to a cell orindirectly via recombination constructs. The endonuclease can beintroduced into a cell transiently or can be incorporated into thegenome of the host cell using any method known in the art. Methods forthe introduction of Cas endonucleases and guide polynucleotide intoplant cells are described, for example, in US 2016/0208272 A1, published21 Jul. 2016, and in US 2016/0201072 A1, published 14 Jul. 2016. In thecase of a CRISPR-Cas system, uptake of the endonuclease and/or theguided polynucleotide into the cell can be facilitated with a CellPenetrating Peptide (CPP) as described in WO2016073433 published May 12,2016.

As used herein, a “genomic region” is a segment of a chromosome in thegenome of a cell that is present on either side of the target site or,alternatively, also comprises a portion of the target site. The genomicregion can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40,5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100,5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100,5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000,5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900,5-3000, 5-3100 or more bases such that the genomic region has sufficienthomology to undergo homologous recombination with the correspondingregion of homology.

TAL effector nucleases (TALEN) are a class of sequence-specificnucleases that can be used to make double-strand breaks at specifictarget sequences in the genome of a plant or other organism. (Miller etal. (2011) Nature Biotechnology 29:143-148).

Endonucleases are enzymes that cleave the phosphodiester bond within apolynucleotide chain. Endonucleases include restriction endonucleases,which cleave DNA at specific sites without damaging the bases, andmeganucleases, also known as homing endonucleases (HEases), which likerestriction endonucleases, bind and cut at a specific recognition site,however the recognition sites for meganucleases are typically longer,about 18 bp or more (patent application PCT/US12/30061, filed on Mar.22, 2012). Meganucleases have been classified into four families basedon conserved sequence motifs, the families are the LAGLIDADG, GIY-YIG,H-N-H, and His-Cys box families. These motifs participate in thecoordination of metal ions and hydrolysis of phosphodiester bonds.HEases are notable for their long recognition sites, and for toleratingsome sequence polymorphisms in their DNA substrates. The namingconvention for meganuclease is similar to the convention for otherrestriction endonuclease. Meganucleases are also characterized by prefixF-, I-, or PI- for enzymes encoded by free-standing ORFs, introns, andinteins, respectively. One step in the recombination process involvespolynucleotide cleavage at or near the recognition site. The cleavingactivity can be used to produce a double-strand break. For reviews ofsite-specific recombinases and their recognition sites, see, Sauer(1994) Curr Op Biotechnol 5:521-7; and Sadowski (1993) FASEB 7:760-7. Insome examples the recombinase is from the Integrase or Resolvasefamilies.

Zinc finger nucleases (ZFNs) are engineered double-strand break inducingagents comprised of a zinc finger DNA binding domain and adouble-strand-break-inducing agent domain. Recognition site specificityis conferred by the zinc finger domain, which typically comprising two,three, or four zinc fingers, for example having a C2H2 structure,however other zinc finger structures are known and have been engineered.Zinc finger domains are amenable for designing polypeptides whichspecifically bind a selected polynucleotide recognition sequence. ZFNsinclude an engineered DNA-binding zinc finger domain linked to anon-specific endonuclease domain, for example nuclease domain from aType IIs endonuclease such as FokI. Additional functionalities can befused to the zinc-finger binding domain, including transcriptionalactivator domains, transcription repressor domains, and methylases. Insome examples, dimerization of nuclease domain is required for cleavageactivity. Each zinc finger recognizes three consecutive base pairs inthe target DNA. For example, a 3 finger domain recognized a sequence of9 contiguous nucleotides, with a dimerization requirement of thenuclease, two sets of zinc finger triplets are used to bind an 18nucleotide recognition sequence.

Genome editing using DSB-inducing agents, such as Cas9-gRNA complexes,has been described, for example in U.S. Patent Application US2015-0082478 A1, published on Mar. 19, 2015, WO2015/026886 A1, publishedon Feb. 26, 2015, WO2016007347, published on Jan. 14, 2016, andWO201625131, published on Feb. 18, 2016.

The term “Cas gene” herein refers to a gene that is generally coupled,associated or close to, or in the vicinity of flanking CRISPR loci inbacterial systems. The terms “Cas gene”, “CRISPR-associated (Cas) gene”are used interchangeably herein. The term “Cas endonuclease” hereinrefers to a protein encoded by a Cas gene. A Cas endonuclease herein,when in complex with a suitable polynucleotide component, is capable ofrecognizing, binding to, and optionally nicking or cleaving all or partof a specific DNA target sequence. A Cas endonuclease described hereincomprises one or more nuclease domains. Cas endonucleases of thedisclosure includes those having a HNH or HNH-like nuclease domainand/or a RuvC or RuvC-like nuclease domain. A Cas endonuclease of thedisclosure includes a Cas9 protein, a Cpf1 protein, a C2c1 protein, aC2c2 protein, a C2c3 protein, Cas3, Cas 5, Cas7, Cas8, Cas10, orcomplexes of these.

As used herein, the terms “guide polynucleotide/Cas endonucleasecomplex”, “guide polynucleotide/Cas endonuclease system”, “guidepolynucleotide/Cas complex”, “guide polynucleotide/Cas system”, “guidedCas system” are used interchangeably herein and refer to at least oneguide polynucleotide and at least one Cas endonuclease that are capableof forming a complex, wherein said guide polynucleotide/Cas endonucleasecomplex can direct the Cas endonuclease to a DNA target site, enablingthe Cas endonuclease to recognize, bind to, and optionally nick orcleave (introduce a single or double strand break) the DNA target site.A guide polynucleotide/Cas endonuclease complex herein can comprise Casprotein(s) and suitable polynucleotide component(s) of any of the fourknown CRISPR systems (Horvath and Barrangou, 2010, Science 327:167-170)such as a type I, II, or III CRISPR system. A Cas endonuclease unwindsthe DNA duplex at the target sequence and optionally cleaves at leastone DNA strand, as mediated by recognition of the target sequence by apolynucleotide (such as, but not limited to, a crRNA or guide RNA) thatis in complex with the Cas protein. Such recognition and cutting of atarget sequence by a Cas endonuclease typically occurs if the correctprotospacer-adjacent motif (PAM) is located at or adjacent to the 3′ endof the DNA target sequence. Alternatively, a Cas protein herein may lackDNA cleavage or nicking activity, but can still specifically bind to aDNA target sequence when complexed with a suitable RNA component. (Seealso U.S. Patent Application US 2015-0082478 A1, published on Mar. 19,2015 and US 2015-0059010 A1, published on Feb. 26, 2015).

A guide polynucleotide/Cas endonuclease complex can cleave one or bothstrands of a DNA target sequence. A guide polynucleotide/Casendonuclease complex that can cleave both strands of a DNA targetsequence typically comprise a Cas protein that has all of itsendonuclease domains in a functional state (e.g., wild type endonucleasedomains or variants thereof retaining some or all activity in eachendonuclease domain). Non-limiting examples of Cas9 nickases suitablefor use herein are disclosed in U.S. Patent Appl. Publ. No.2014/0189896.

Other Cas endonuclease systems have been described in PCT patentapplications PCT/US16/32073, filed May 12, 2016 and PCT/US16/32028 filedMay 12, 2016.

“Cas9” (formerly referred to as Cas5, Csn1, or Csx12) herein refers to aCas endonuclease of a type II CRISPR system that forms a complex with acrNucleotide and a tracrNucleotide, or with a single guidepolynucleotide, for specifically recognizing and cleaving all or part ofa DNA target sequence. Cas9 protein comprises a RuvC nuclease domain andan HNH (H-N-H) nuclease domain, each of which can cleave a single DNAstrand at a target sequence (the concerted action of both domains leadsto DNA double-strand cleavage, whereas activity of one domain leads to anick). In general, the RuvC domain comprises subdomains I, II and III,where domain I is located near the N-terminus of Cas9 and subdomains IIand III are located in the middle of the protein, flanking the HNHdomain (Hsu et al, Cell 157:1262-1278). A type II CRISPR system includesa DNA cleavage system utilizing a Cas9 endonuclease in complex with atleast one polynucleotide component. For example, a Cas9 can be incomplex with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA(tracrRNA). In another example, a Cas9 can be in complex with a singleguide RNA.

Any guided endonuclease can be used in the methods disclosed herein.Such endonucleases include, but are not limited to Cas9 and Cpf1endonucleases. Many endonucleases have been described to date that canrecognize specific PAM sequences (see for example—Jinek et al. (2012)Science 337 p 816-821, PCT patent applications PCT/US16/32073, filed May12, 2016 and PCT/US16/32028 filed May 12, 2016 and Zetsche B et al.2015. Cell 163, 1013) and cleave the target DNA at a specific position.It is understood that based on the methods and embodiments describedherein utilizing a guided Cas system one can now tailor these methodssuch that they can utilize any guided endonuclease system.

The guide polynucleotide can also be a single molecule (also referred toas single guide polynucleotide) comprising a crNucleotide sequencelinked to a tracrNucleotide sequence. The single guide polynucleotidecomprises a first nucleotide sequence domain (referred to as VariableTargeting domain or VT domain) that can hybridize to a nucleotidesequence in a target DNA and a Cas endonuclease recognition domain (CERdomain), that interacts with a Cas endonuclease polypeptide. By “domain”it is meant a contiguous stretch of nucleotides that can be RNA, DNA,and/or RNA-DNA-combination sequence. The VT domain and/or the CER domainof a single guide polynucleotide can comprise a RNA sequence, a DNAsequence, or a RNA-DNA-combination sequence. The single guidepolynucleotide being comprised of sequences from the crNucleotide andthe tracrNucleotide may be referred to as “single guide RNA” (whencomposed of a contiguous stretch of RNA nucleotides) or “single guideDNA” (when composed of a contiguous stretch of DNA nucleotides) or“single guide RNA-DNA” (when composed of a combination of RNA and DNAnucleotides). The single guide polynucleotide can form a complex with aCas endonuclease, wherein said guide polynucleotide/Cas endonucleasecomplex (also referred to as a guide polynucleotide/Cas endonucleasesystem) can direct the Cas endonuclease to a genomic target site,enabling the Cas endonuclease to recognize, bind to, and optionally nickor cleave (introduce a single or double strand break) the target site.(See also U.S. Patent Application US 2015-0082478 A1, published on Mar.19, 2015 and US 2015-0059010 A1, published on Feb. 26, 2015.)

The term “variable targeting domain” or “VT domain” is usedinterchangeably herein and includes a nucleotide sequence that canhybridize (is complementary) to one strand (nucleotide sequence) of adouble strand DNA target site. In some embodiments, the variabletargeting domain comprises a contiguous stretch of 12 to 30 nucleotides.The variable targeting domain can be composed of a DNA sequence, a RNAsequence, a modified DNA sequence, a modified RNA sequence, or anycombination thereof.

The terms “single guide RNA” and “sgRNA” are used interchangeably hereinand relate to a synthetic fusion of two RNA molecules, a crRNA (CRISPRRNA) comprising a variable targeting domain (linked to a tracr matesequence that hybridizes to a tracrRNA), fused to a tracrRNA(trans-activating CRISPR RNA). The single guide RNA can comprise a crRNAor crRNA fragment and a tracrRNA or tracrRNA fragment of the type IICRISPR/Cas system that can form a complex with a type II Casendonuclease, wherein said guide RNA/Cas endonuclease complex can directthe Cas endonuclease to a DNA target site, enabling the Cas endonucleaseto recognize, bind to, and optionally nick or cleave (introduce a singleor double strand break) the DNA target site.

The terms “guide RNA/Cas endonuclease complex”, “guide RNA/Casendonuclease system”, “guide RNA/Cas complex”, “guide RNA/Cas system”,“gRNA/Cas complex”, “gRNA/Cas system”, “RNA-guided endonuclease”, “RGEN”are used interchangeably herein and refer to at least one RNA componentand at least one Cas endonuclease that are capable of forming a complex,wherein said guide RNA/Cas endonuclease complex can direct the Casendonuclease to a DNA target site, enabling the Cas endonuclease torecognize, bind to, and optionally nick or cleave (introduce a single ordouble strand break) the DNA target site. A guide RNA/Cas endonucleasecomplex herein can comprise Cas protein(s) and suitable RNA component(s)of any of the four known CRISPR systems (Horvath and Barrangou, 2010,Science 327:167-170) such as a type I, II, or III CRISPR system. A guideRNA/Cas endonuclease complex can comprise a Type II Cas9 endonucleaseand at least one RNA component (e.g., a crRNA and tracrRNA, or a gRNA).(See also U.S. Patent Application US 2015-0082478 A1, published on Mar.19, 2015 and US 2015-0059010 A1, published on Feb. 26, 2015).

The guide polynucleotide can be introduced into a cell transiently, assingle stranded polynucleotide or a double stranded polynucleotide,using any method known in the art such as, but not limited to, particlebombardment, Agrobacterium transformation or topical applications. Theguide polynucleotide can also be introduced indirectly into a cell byintroducing a recombinant DNA molecule (via methods such as, but notlimited to, particle bombardment or Agrobacterium transformation)comprising a heterologous nucleic acid fragment encoding a guidepolynucleotide, operably linked to a specific promoter that is capableof transcribing the guide RNA in said cell. The specific promoter canbe, but is not limited to, a RNA polymerase III promoter, which allowfor transcription of RNA with precisely defined, unmodified, 5′- and3′-ends (DiCarlo et al., Nucleic Acids Res. 41: 4336-4343; Ma et al.,Mol. Ther. Nucleic Acids 3:e161) as described in WO2016025131, publishedon Feb. 18, 2016.

The terms “target site”, “target sequence”, “target site sequence,“target DNA”, “target locus”, “genomic target site”, “genomic targetsequence”, “genomic target locus” and “protospacer”, are usedinterchangeably herein and refer to a polynucleotide sequence such as,but not limited to, a nucleotide sequence on a chromosome, episome, orany other DNA molecule in the genome (including chromosomal,chloroplastic, mitochondrial DNA, plasmid DNA) of a cell, at which aguide polynucleotide/Cas endonuclease complex can recognize, bind to,and optionally nick or cleave. The target site can be an endogenous sitein the genome of a cell, or alternatively, the target site can beheterologous to the cell and thereby not be naturally occurring in thegenome of the cell, or the target site can be found in a heterologousgenomic location compared to where it occurs in nature. As used herein,terms “endogenous target sequence” and “native target sequence” are usedinterchangeable herein to refer to a target sequence that is endogenousor native to the genome of a cell and is at the endogenous or nativeposition of that target sequence in the genome of the cell. Cellsinclude, but are not limited to, human, non-human, animal, bacterial,fungal, insect, yeast, non-conventional yeast, and plant cells as wellas plants and seeds produced by the methods described herein. An“artificial target site” or “artificial target sequence” are usedinterchangeably herein and refer to a target sequence that has beenintroduced into the genome of a cell. Such an artificial target sequencecan be identical in sequence to an endogenous or native target sequencein the genome of a cell but be located in a different position (i.e., anon-endogenous or non-native position) in the genome of a cell.

Provided are plants and seeds which contain an altered or modifiedtarget site or sequence. An “altered target site”, “altered targetsequence”, “modified target site”, “modified target sequence” are usedinterchangeably herein and refer to a target sequence as disclosedherein that comprises at least one alteration when compared tonon-altered target sequence. Such “alterations” include, for example:(i) replacement of at least one nucleotide, (ii) a deletion of at leastone nucleotide, (iii) an insertion of at least one nucleotide, or (iv)any combination of (i)-(iii).

Methods for “modifying a target site” and “altering a target site” areused interchangeably herein and refer to methods for producing analtered target site.

The length of the target DNA sequence (target site) can vary, andincludes, for example, target sites that are at least 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morenucleotides in length. It is further possible that the target site canbe palindromic, that is, the sequence on one strand reads the same inthe opposite direction on the complementary strand. The nick/cleavagesite can be within the target sequence or the nick/cleavage site couldbe outside of the target sequence. In another variation, the cleavagecould occur at nucleotide positions immediately opposite each other toproduce a blunt end cut or, in other Cases, the incisions could bestaggered to produce single-stranded overhangs, also called “stickyends”, which can be either 5′ overhangs, or 3′ overhangs. Activevariants of genomic target sites can also be used. Such active variantscan comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to the given targetsite, wherein the active variants retain biological activity and henceare capable of being recognized and cleaved by an Cas endonuclease.Assays to measure the single or double-strand break of a target site byan endonuclease are known in the art and generally measure the overallactivity and specificity of the agent on DNA substrates containingrecognition sites.

A “protospacer adjacent motif” (PAM) herein refers to a short nucleotidesequence adjacent to a target sequence (protospacer) that is recognized(targeted) by a guide polynucleotide/Cas endonuclease system describedherein. The Cas endonuclease may not successfully recognize a target DNAsequence if the target DNA sequence is not followed by a PAM sequence.The sequence and length of a PAM herein can differ depending on the Casprotein or Cas protein complex used. The PAM sequence can be of anylength but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 nucleotides long.

The terms “targeting”, “gene targeting” and “DNA targeting” are usedinterchangeably herein. DNA targeting herein may be the specificintroduction of a knock-out, edit, or knock-in at a particular DNAsequence, such as in a chromosome or plasmid of a cell. In general, DNAtargeting can be performed herein by cleaving one or both strands at aspecific DNA sequence in a cell with an endonuclease associated with asuitable polynucleotide component. Such DNA cleavage, if a double-strandbreak (DSB), can prompt NHEJ or HDR processes which can lead tomodifications at the target site.

A targeting method herein can be performed in such a way that two ormore DNA target sites are targeted in the method, for example. Such amethod can optionally be characterized as a multiplex method. Two,three, four, five, six, seven, eight, nine, ten, or more target sitescan be targeted at the same time in certain embodiments. A multiplexmethod is typically performed by a targeting method herein in whichmultiple different RNA components are provided, each designed to guide aguide polynucleotide/Cas endonuclease complex to a unique DNA targetsite.

Provided are plants and seeds in which a functional sequence has beenknocked out. The terms “knock-out”, “gene knock-out” and “geneticknock-out” are used interchangeably herein. A knock-out represents a DNAsequence of a cell that has been rendered partially or completelyinoperative by targeting with a Cas protein; such a DNA sequence priorto knock-out could have encoded an amino acid sequence, or could havehad a regulatory function (e.g., promoter), for example. A knock-out maybe produced by an indel (insertion or deletion of nucleotide bases in atarget DNA sequence through NHEJ), or by specific removal of sequencethat reduces or completely destroys the function of sequence at or nearthe targeting site.

In some aspects, the guide polynucleotide/Cas endonuclease system can beused in combination with a co-delivered polynucleotide modificationtemplate to allow for editing (modification) of a genomic nucleotidesequence of interest (see also U.S. Patent Application US 2015-0082478A1, published on Mar. 19, 2015 and WO2015/026886 A1, published on Feb.26, 2015), or co-delivered with a heterologous (donor) DNA molecule forintegration at the double-strand break via homologous recombination.

Provided are plants and seeds in which a functional sequence has beenknocked in. The terms “knock-in”, “gene knock-in, “gene insertion” and“genetic knock-in” are used interchangeably herein. A knock-inrepresents the replacement or insertion of a DNA sequence at a specificDNA sequence in cell by targeting with a Cas protein (by HR, wherein asuitable donor DNA polynucleotide is also used). Examples of knock-insare a specific insertion of a heterologous amino acid coding sequence ina coding region of a gene, or a specific insertion of a transcriptionalregulatory element in a genetic locus.

Various methods and compositions can be employed to obtain a cell ororganism having a polynucleotide of interest inserted in a target sitefor a Cas endonuclease. Such methods can employ homologous recombinationto provide integration of the polynucleotide of Interest at the targetsite. In one method provided, a polynucleotide of interest is providedto the organism cell in a donor DNA construct. As used herein, “donorDNA” is a DNA construct that comprises a polynucleotide of Interest tobe inserted into the target site of a Cas endonuclease. The donor DNAconstruct further comprises a first and a second region of homology thatflank the polynucleotide of Interest. The first and second regions ofhomology of the donor DNA share homology to a first and a second genomicregion, respectively, present in or flanking the target site of the cellor organism genome. By “homology” is meant DNA sequences that aresimilar. For example, a “region of homology to a genomic region” that isfound on the donor DNA is a region of DNA that has a similar sequence toa given “genomic region” in the cell or organism genome. A region ofhomology can be of any length that is sufficient to promote homologousrecombination at the cleaved target site. For example, the region ofhomology can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40,5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100,5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100,5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000,5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900,5-3000, 5-3100 or more bases in length such that the region of homologyhas sufficient homology to undergo homologous recombination with thecorresponding genomic region. “Sufficient homology” indicates that twopolynucleotide sequences have sufficient structural similarity to act assubstrates for a homologous recombination reaction. The structuralsimilarity includes overall length of each polynucleotide fragment, aswell as the sequence similarity of the polynucleotides. Sequencesimilarity can be described by the percent sequence identity over thewhole length of the sequences, and/or by conserved regions comprisinglocalized similarities such as contiguous nucleotides having 100%sequence identity, and percent sequence identity over a portion of thelength of the sequences.

The amount of sequence identity shared by a target and a donorpolynucleotide can vary and includes total lengths and/or regions havingunit integer values in the ranges of about 1-20 bp, 20-50 bp, 50-100 bp,75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp,350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total lengthof the target site. These ranges include every integer within the range,for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bps. The amount ofhomology can also be described by percent sequence identity over thefull aligned length of the two polynucleotides which includes percentsequence identity of at least or at least about 50%, 55%, 60%, 65%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% and less than or less than about 100%, 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91% or 90%. Sufficient homology includes anycombination of polynucleotide length, global percent sequence identity,and optionally conserved regions of contiguous nucleotides or localpercent sequence identity, for example sufficient homology can bedescribed as a region of 75-150 bp having at least 80% sequence identityto a region of the target locus. Sufficient homology can also bedescribed by the predicted ability of two polynucleotides tospecifically hybridize under high stringency conditions, see, forexample, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, NY); Current Protocols inMolecular Biology, Ausubel et al., Eds (1994) Current Protocols, (GreenePublishing Associates, Inc. and John Wiley & Sons, Inc.); and, Tijssen(1993) Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, (Elsevier, New York).

The structural similarity between a given genomic region and thecorresponding region of homology found on the donor DNA can be anydegree of sequence identity that allows for homologous recombination tooccur. For example, the amount of homology or sequence identity sharedby the “region of homology” of the donor DNA and the “genomic region” ofthe organism genome can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that thesequences undergo homologous recombination

The region of homology on the donor DNA can have homology to anysequence flanking the target site. While in some embodiments the regionsof homology share significant sequence homology to the genomic sequenceimmediately flanking the target site, it is recognized that the regionsof homology can be designed to have sufficient homology to regions thatmay be further 5′ or 3′ to the target site. In still other embodiments,the regions of homology can also have homology with a fragment of thetarget site along with downstream genomic regions. In one embodiment,the first region of homology further comprises a first fragment of thetarget site and the second region of homology comprises a secondfragment of the target site, wherein the first and second fragments aredissimilar.

As used herein, “homologous recombination” includes the exchange of DNAfragments between two DNA molecules at the sites of homology.

Further uses for guide RNA/Cas endonuclease systems have been described(See U.S. Patent Application US 2015-0082478 A1, published on Mar. 19,2015, WO2015/026886 A1, published on Feb. 26, 2015, US 2015-0059010 A1,published on Feb. 26, 2015, PCT application publication WO2016007347A1,published 14 Jan. 2016, and PCT application publication WO2016025131A1,published 18 Feb. 2016) and include but are not limited to modifying orreplacing nucleotide sequences of interest (such as a regulatoryelements), insertion of polynucleotides of interest, gene knock-out,gene-knock in, modification of splicing sites and/or introducingalternate splicing sites, modifications of nucleotide sequences encodinga protein of interest, amino acid and/or protein fusions, and genesilencing by expressing an inverted repeat into a gene of interest.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published, amongothers, for cotton (U.S. Pat. Nos. 5,004,863, 5,159,135); soybean (U.S.Pat. Nos. 5,569,834, 5,416,011); Brassica (U.S. Pat. No. 5,463,174);peanut (Cheng et al., Plant Cell Rep. 15:653-657 (1996), McKently etal., Plant Cell Rep. 14:699-703 (1995)); papaya (Ling et al.,Bio/technology 9:752-758 (1991)); and pea (Grant et al., Plant Cell Rep.15:254-258 (1995)). For a review of other commonly used methods of planttransformation see Newell, C. A., Mol. Biotechnol. 16:53-65 (2000). Oneof these methods of transformation uses Agrobacterium rhizogenes(Tepfler, M. and Casse-Delbart, F., Microbiol. Sci. 4:24-28 (1987)).Transformation of soybeans using direct delivery of DNA has beenpublished using PEG fusion (PCT Publication No. WO 92/17598),electroporation (Chowrira et al., Mol. Biotechnol. 3:17-23 (1995);Christou et al., Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966 (1987)),microinjection, or particle bombardment (McCabe et al., Biotechnology6:923-926 (1988); Christou et al., Plant Physiol. 87:671-674 (1988)).

There are a variety of methods for the regeneration of plants from planttissues. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, Eds.; InMethods for Plant Molecular Biology; Academic Press, Inc.: San Diego,Calif., 1988). This regeneration and growth process typically includesthe steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentor through the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent disclosure containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

This disclosure also concerns a method of decreasing the expression ofat least one nucleic acid such as a heterologous nucleic acid fragmentin a plant cell which comprises:

-   -   (a) transforming a plant cell with the recombinant expression        construct described herein;    -   (b) growing fertile mature plants from the transformed plant        cell of step (a);    -   (c) selecting plants containing a transformed plant cell wherein        the expression of the nucleic acid such as a heterologous        nucleic acid fragment is increased or decreased.

Transformation and selection can be accomplished using methodswell-known to those skilled in the art including, but not limited to,the methods described herein.

The soybean seeds can be processed to produce oil and protein. Methodsof processing the soybean seeds to produce oil and protein are providedwhich include one or more steps of dehulling the seeds, crushing theseeds, heating the seeds, such as with steam, extracting the oil,roasting, and extrusion. Processing and oil extraction can be done usingsolvents or mechanical extraction.

Products formed following processing include, without limitation, soynuts, soy milk, tofu, texturized soy protein, soybean oil, soy proteinflakes, isolated soy protein. Crude or partially degummed oil can befurther processed by one or more of degumming, alkali treatment, silicaabsorption, vacuum bleaching, hydrogenation, interesterification,filtration, deodorization, physical refining, refractionation, andoptional blending to produce refined bleached deodorized (RBD) oil.

The oil and protein can be used in animal feed and in food products forhuman consumption. Provided are food products and animal feed comprisingoils, protein and compositions described herein. The food products andanimal feed may comprise nucleotides comprising one or more of themodified alleles disclosed herein.

Methods of detecting the modified polynucleotides are provided. Methodsof extracting modified DNA from a sample or detecting the presence ofDNA corresponding to the modified genomic sequences comprising deletionsof FAD2-1 and FAD3, such as presented in FIGS. 1-5 can be carried out.Such methods comprise contacting a sample comprising soybean genomic DNAwith a DNA primer set, that when used in a nucleic acid amplificationreaction, such as the polymerase chain reaction (PCR), with genomic DNAextracted from soybeans produces an amplicon that is diagnostic foreither the presence or absence of the modified FAD2-1A and FAD3 alleles.The methods include the steps of performing a nucleic acid amplificationreaction, thereby producing the amplicon and detecting the amplicon.

In some embodiments one of the pair of DNA molecules comprises the wildtype sequence where the modification occurs with the second of the pairbeing upstream or downstream as appropriate and suitably in proximity tothe wild type sequence where the modification occurs, such that anamplicon is produced when the wild type allele is present, but noamplicon is produced when the modified allele is present. Suitableprimers and probes for use in reactions to detect the presence of thealleles of FAD2-1A (e.g. SEQ ID NOs: 10, 11 and 12 and functionalfragments thereof), FAD2-1B (e.g. SEQ ID NOs: 13, 11 and 14 andfunctional fragments thereof), FAD3a (e.g. SEQ ID NOs: 15, 16 and 17 andfunctional fragments thereof) and FAD3b (e.g. SEQ ID NOs: 18, 16 and 17and functional fragments thereof) are provided in Table 2 and describedin Example 4. In the context of the methods, in proximity meanssufficiently close such that the distance between the first and secondof the pair of DNA molecules facilitates the production of an ampliconwhen included in a DNA amplification reaction comprising soybean genomicDNA. For example, the second primer may bind at a location beginning at,within or less than 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500,16, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700,2800, 2900, 3000, 3500, 4000, 4500 or 5000 nucleotides upstream ordownstream of the end of the binding site of the first DNA primermolecule.

Probes and primers are provided which are of sufficient nucleotidelength to bind specifically to the target DNA sequence under thereaction or hybridization conditions. Suitable probes and primers are atleast 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 nucleotides in length, and less than 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, or 12 nucleotides in length. Such probes and primers canhybridize specifically to a target sequence under high stringencyhybridization conditions. Preferably, probes and primers have completeor 100% DNA sequence similarity of contiguous nucleotides with thetarget sequence, although probes which differ from the target DNAsequence but retain the ability to hybridize to target DNA sequence maybe also be used. Reverse complements of the primers and probes disclosedherein are also provided and can be used in the methods and compositionsdescribed herein.

In some embodiments, one of the pair of DNA molecules comprises themodification or traverses the modification junction such as the deletionjunctions depicted in FIGS. 1-5, with the second DNA molecule of thepair being upstream or downstream of the genomic sequence asappropriate, such that an amplicon is produced when the modified alleleis present, but no amplicon is produced when the wild type allele ispresent. Suitable primers for use in reactions to detect the presence ofthe modified alleles can be designed based on the junction sequencesdepicted in FIGS. 1-5 for the modified alleles.

For example, for SEQ ID NO: 54, the deletion junction occurs betweenpositions 27 and 28; a primer can be designed which begins (or includesif beginning before position 1) at position 1 through position 27 andends (or includes if ending after position 55) at position 29 to 55,provided that the primer is of sufficient length to function in theamplification reaction. The reverse complement of such a primer is alsoprovided.

For example, for SEQ ID NO: 55, the deletion junction occurs betweenpositions 23 and 24; a primer can be designed which begins (or includesif beginning before position 1) at position 1 through position 23 andends (or includes if ending after position 55) at position 24 to 55,provided that the primer is of sufficient length to function in theamplification reaction. The reverse complement of such a primer is alsoprovided.

For example, for SEQ ID NO: 57, the deletion junction occurs betweenpositions 27 and 28; a primer can be designed which begins (or includesif beginning before position 1) at position 1 through position 27 andends (or includes if ending after position 57) at position 28 to 57,provided that the primer is of sufficient length to function in theamplification reaction. The reverse complement of such a primer is alsoprovided.

For example, for SEQ ID NO: 58, the deletion junction occurs betweenpositions 27 and 28; a primer can be designed which begins (or includesif beginning before position 1) at position 1 through position 27 andends (or includes if ending after position 55) at position 29 to 55,provided that the primer is of sufficient length to function in theamplification reaction. The reverse complement of such a primer is alsoprovided.

For example, for SEQ ID NO: 60, the deletion junction occurs betweenpositions 20 and 21; a primer can be designed which begins (or includesif beginning before position 1) at position 1 through position 20 andends (or includes if ending after position 60) at position 22 to 60,provided that the primer is of sufficient length to function in theamplification reaction. The reverse complement of such a primer is alsoprovided.

For example, for SEQ ID NO: 61, the deletion junction occurs betweenpositions 20 and 21; a primer can be designed which begins (or includesif beginning before position 1) at position 1 through position 20 andends (or includes if ending after position 58) at position 21 to 58,provided that the primer is of sufficient length to function in theamplification reaction. The reverse complement of such a primer is alsoprovided.

For example, for SEQ ID NOs: 63 and 64, the deletion junction occursbetween positions 20 and 21; a primer can be designed which begins (orincludes if beginning before position 1) at position 1 through position20 and ends (or includes if ending after position 60) at position 22 to60, provided that the primer is of sufficient length to function in theamplification reaction. The reverse complement of such a primer is alsoprovided.

According to another aspect of the invention, methods of detecting thepresence of a DNA molecule corresponding to the modified FAD2-1 and FAD3alleles in a sample, include contacting the sample comprising DNAextracted from a soybean plant, cell or seed with a DNA probe moleculethat hybridizes under stringent hybridization conditions with genomicDNA from a soybean comprising the modified FAD2-1 or FAD3 alleles anddoes not hybridize under stringent hybridization conditions with acontrol soybean plant DNA, The sample and probe are subjected tostringent hybridization conditions and hybridization of the probe to theDNA from the soybean plant, cell or seed comprising the modified FAD2-1or FAD3

In some embodiments the primers and probes bind and traverse themodification, such as a deletion junction, in the genomic DNA and have asequence following the modification or junction in the 5′ to 3′direction which is less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotidesin length and at least 1, 2, 3, 4 or 5 nucleotides in length.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating embodiments of the invention, are given by way ofillustration only. From the above discussion and these Examples, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions. Such modifications are also intended tofall within the scope of the appended claims. For example, one of skillin the art could use template directed repair, insertion of aheterologous polynucleotide to disrupt the native gene, or modificationusing a deaminase to alter a base to create the same or similar edits tothose disclosed in these examples.

Example 1 Soybean Optimized Expression Cassettes for Guide RNA/CasEndonuclease Based Genome Modification in Soybean Plants

For genome engineering applications, the type II CRISPR/Cas systemminimally requires the Cas9 protein and a duplexed crRNA/tracrRNAmolecule or a synthetically fused crRNA and tracrRNA (guide RNA)molecule for DNA target site recognition and cleavage (Gasiunas et al.(2012) Proc. Natl. Acad. Sci. USA 109: E2579-86, Jinek et al. (2012)Science 337:816-21, Mali et al. (2013) Science 339:823-26, and Cong etal. (2013) Science 339:819-23). Described herein is a guideRNA/Casendonuclease system that is based on the type II CRISPR/Cas system andconsists of a Cas endonuclease and a guide RNA (or duplexed crRNA andtracrRNA) that together can form a complex that recognizes a genomictarget site in a plant and introduces a double-strand -break into saidtarget site.

To use the guide RNA/Cas endonuclease system in soybean, the Cas9 genefrom Streptococcus pyogenes M1 GAS (SF370) was soybean codon optimized(SEQ ID NO: 1) per standard techniques known in the art. To facilitatenuclear localization of the Cas9 protein in soybean cells, Simian virus40 (SV40) monopartite amino terminal nuclear localization signal(MAPKKKRKV, SEQ ID NO: 2) and Agrobacterium tumefaciens bipartite VirD2T-DNA border endonuclease carboxyl terminal nuclear localization signal(KRPRDRHDGELGGRKRAR, SEQ ID NO: 3) were incorporated at the amino andcarboxyl-termini of the Cas9 open reading frame, respectively. Thesoybean optimized Cas9 gene was operably linked to a soybeanconstitutive promoter such as the strong soybean constitutive promoterGM-EF1A2 (US patent application 20090133159 (SEQ ID NO: 4). or regulatedpromoter by standard molecular biological techniques.

The second component necessary to form a functional guide RNA/Casendonuclease system for genome engineering applications is a duplex ofthe crRNA and tracrRNA molecules or a synthetic fusing of the crRNA andtracrRNA molecules, a guide RNA. To confer efficient guide RNAexpression (or expression of the duplexed crRNA and tracrRNA) insoybean, the soybean U6 polymerase III promoter and U6 polymerase IIIterminator were used.

Plant U6 RNA polymerase III promoters have been cloned and characterizedfrom such as Arabidopsis and Medicago truncatula (Waibel and Filipowicz,NAR 18:3451-3458 (1990); Li et al., J. Integrat. Plant Biol. 49:222-229(2007); Kim and Nam, Plant Mol. Biol. Rep. 31:581-593 (2013); Wang etal., RNA 14:903-913 (2008)). Soybean U6 small nuclear RNA (snRNA) geneswere identified herein by searching public soybean variety Williams82genomic sequence using Arabidopsis U6 gene coding sequence.Approximately 0.5 kb genomic DNA sequence upstream of the first Gnucleotide of a U6 gene was selected to be used as a RNA polymerase IIIpromoter for example, GM-U6-13.1 promoter (SEQ ID NO:5), to expressguide RNA to direct Cas9 nuclease to designated genomic site. The guideRNA coding sequence was 76 bp long and comprised a 20 bp variabletargeting domain from a chosen soybean genomic target site on the 5′ endand a tract of 4 or more T residues as a transcription terminator on the3′ end. The first nucleotide of the 20 bp variable targeting domain wasa G residue to be used by RNA polymerase III for transcription. Othersoybean U6 homologous genes promoters were similarly cloned and used forsmall RNA expression.

Since the Cas9 endonuclease and the guide RNA need to form a protein/RNAcomplex to mediate site-specific DNA double strand cleavage, the Cas9endonuclease and guide RNA must be expressed in same cells. To improvetheir co-expression and presence, the Cas9 endonuclease and guide RNAexpression cassettes were linked into a single DNA construct.

Example 2 Selection of Soybean FAD2 and FAD3 Target Sites to be Cleavedby the Guide RNA/Cas Endonuclease System

A. guideRNA/Cas9 Endonuclease Target Site Design on the Soybean FAD2-1and FAD3 Genes

There are two seed-preferred FAD2-1 genes in soybean (FAD2-1A forGlyma.10g278000 and FAD2-1B for Glyma.20g111000). One guide RNA/Cas9endonuclease target site (GM-FAD2-1 CR1) was designed to target both theFAD2-1 genes (Table 1). There are also two major FAD3 genes in soybean(FAD3a for Glyma.14g194300 and FAD3b for Glyma.02g227200). The GM-FAD3CR2 site was designed to target both FAD3 genes (Table 1)

TABLE 1 Guide RNA/Cas9 endonuclease target sites on soybean FAD2-1 genesand FAD3 genes Cas endonuclease Name of target gRNA-Cas9 sequenceendonuclease (SEQ target site ID NO:) Physical location GM-FAD2-1 6Gm10: 50014185..50014166 CR1 Gm20: 35317773..35317754 GM-FAD3 7 Gm14:45939600..445939618 CR2 Gm02: 41423563..41423581B. Guide-RNA Expression Cassettes, Cas9 Endonuclease ExpressionCassettes and Knockout of the Soybean FAD2-1 and FAD3 Genes.

The soybean U6 small nuclear RNA promoter, GM-U6-13.1 (SEQ ID NO: 5),was used to express guide RNAs to direct Cas9 nuclease to designatedgenomic target sites. A soybean codon optimized Cas9 endonuclease (SEQID NO: 1) expression cassette and a guide RNA expression cassette werelinked in the plasmid (RTW1211 or RTW1312). For examples, the RTW1211construct (SEQ ID NO.8), which contained the GM-FAD2-1 CR1 gRNAexpression cassette and the cas9 expression cassette, was made to targetboth the FAD2-1A and FAD2-1B genes simultaneously. Similarly, theRTW1312 construct (SEQ ID NO.9) was made to target both the FAD3a andFad3b genes at the same time.

Example 3 Delivery of the Guide RNA/Cas9 Endonuclease System DNA toSoybean by Stable Transformation

Soybean somatic embryogenic suspension cultures were induced from aDuPont Pioneer proprietary elite cultivar 93Y21 as follows. Cotyledons(˜3 mm in length) were dissected from surface sterilized, immature seedsand were cultured for 6-10 weeks in the light at 26° C. on a Murashigeand Skoog (MS) media containing 0.7% agar and supplemented with 10 mg/ml2,4-D (2,4-Dichlorophenoxyacetic acid). Globular stage somatic embryos,which produced secondary embryos, were then excised and placed intoflasks containing liquid MS medium supplemented with 2,4-D (10 mg/ml)and cultured in light on a rotary shaker. After repeated selection forclusters of somatic embryos that multiplied as early, globular stagedembryos, the soybean embryogenic suspension cultures were maintained in35 ml liquid media on a rotary shaker, 150 rpm, at 26° C. withfluorescent lights on a 16:8-hour day/night schedule. Cultures weresubcultured every two weeks by inoculating approximately 35 mg of tissueinto 35 ml of the same fresh liquid MS medium.

Soybean embryogenic suspension cultures were then transformed by themethod of particle gun bombardment using a DuPont Biolistic™ PDS1000/HEinstrument (Bio-Rad Laboratories, Hercules, Calif.). To 50 μl of a 60mg/ml 1.0 mm gold particle suspension were added in order: 30 μl ofequal amount (30 ng/μl) plasmid DNA, 20 μl of 0.1 M spermidine, and 25μl of 5 M CaCl₂. The particle preparation was then agitated for 3minutes, spun in a centrifuge for 10 seconds and the supernatantremoved. The DNA-coated particles were then washed once in 400 μl 100%ethanol and resuspended in 45 μl of 100% ethanol. The DNA/particlesuspension was sonicated three times for one second each. Then 5 μl ofthe DNA-coated gold particles was loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture was placedin an empty 60×15 mm Petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5 to 10 plates of tissue were bombarded. Membrane rupture pressure wasset at 1100 psi and the chamber was evacuated to a vacuum of 28 inchesmercury. The tissue was placed approximately 3.5 inches away from theretaining screen and bombarded once. Following bombardment, the tissuewas divided in half and placed back into liquid media and cultured asdescribed above.

Five to seven days post bombardment, the liquid media was exchanged withfresh media containing 30 mg/ml hygromycin as selection agent. Thisselective media was refreshed weekly. Seven to eight weeks postbombardment, green, transformed tissue was observed growing fromuntransformed, necrotic embryogenic clusters. Isolated green tissue wasremoved and inoculated into individual flasks to generate new, clonallypropagated, transformed embryogenic suspension cultures. Each clonallypropagated culture was treated as an independent transformation eventand subcultured in the same liquid MS media supplemented with 2,4-D (10mg/ml) and 30 ng/ml hygromycin selection agent to increase mass. Theembryogenic suspension cultures were then transferred to agar solid MSmedia plates without 2,4-D supplement to allow somatic embryos todevelop. A sample of each event was collected at this stage forquantitative PCR analysis.

Cotyledon stage somatic embryos were dried-down (by transferring theminto an empty small Petri dish that was seated on top of a 10 cm Petridish containing some agar gel to allow slow dry down) to mimic the laststages of soybean seed development. Dried-down embryos were placed ongermination solid media and transgenic soybean plantlets wereregenerated. The transgenic plants were then transferred to soil andmaintained in growth chambers for seed production. Transgenic eventswere sampled at somatic embryo stage or T0 leaf stage for molecularanalysis.

Example 4 Detection of Site-Specific Mutations with NHEJ Mediated by theGuide RNA/Cas9 System in Stably Transformed Soybean

Genomic DNA was extracted from somatic embryo samples and analyzed byquantitative PCR using a 7500 real time PCR system (Applied Biosystems,Foster City, Calif.) with target site-specific primers and FAM-labeledfluorescence probe to check copy number changes of the target sites. TheqPCR analysis was done in duplex reactions with a syringolide inducedprotein (SIP) gene as the endogenous controls and a wild type 93Y21genomic DNA sample that contains one copy of the target site with 2alleles, as the single copy calibrator. The endogenous control probeSIP-T was labeled with VIC and the gene-specific probes FAD2-T1, FAD2-T2and FAD3-T2 were labeled with FAM (Table 2) for the simultaneousdetection of both fluorescent probes (Applied Biosystems). PCR reactiondata were captured and analyzed using the sequence detection softwareprovided with the 7500 real time PCR system and the gene copy numberswere calculated using the relative quantification methodology (AppliedBiosystems).

TABLE 2 Primers/probes used in qPCR analyses of transgenic soybeanevents. Target Primer/Probe SEQ ID Site Name Sequences NOs: FAD2-1AFAD2-F1 TCGTGTGGCCAAAGTGGAA 10 FAD2-R1 TTTGTGTTTGGAACCCTTGAGA 11 FAD2-T1TTCAAGGGAAGAAGCC 12 (FAM-MGB) FAD2-1B FAD2-F2 CCGTGTGGCCAAAGTTGAA 13FAD2-R1 TTTGTGTTTGGAACCCTTGAGA 11 FAD2-T2 TTCAGCAGAAGAAGCC 14 (FAM-MGB)FAD3a FAD3-F1 TAATGGATACCAAAAGGAAGC 15 FAD3-R2 CAAGCACATCCCTGAGAACATAAC16 FAD3-T2 AATCCATGGAGATCCCT 17 (FAM-MGB) FAD3b FAD3-F2ATACCAACAAAAGGGTTCTTC 18 FAD3-R2 CAAGCACATCCCTGAGAACATAAC 16 FAD3-T2AATCCATGGAGATCCCT 17 (FAM-MGB) CAS9 Cas9-F CCTTCTTCCACCGCCTTGA 19 Cas9-RTGGGTGTCTCTCGTGCTTTTT 20 Cas9-T AATCATTCCTGGTGGAGGA 21 (FAM-MGB) pINIIpINII-99F TGATGCCCACATTATAGTGATTAGC 22 pINII-13R CATCTTCTGGATTGGCCAACTT23 pINII-69T ACTATGTGTGCATCCTT 24 (FAM-MGB) SIP SIP-130FTTCAAGTTGGGCTTTTTCAGAAG 25 SIP-198R TCTCCTTGGTGCTCTCATCACA 26 SIP-170TCTGCAGCAGAACCAA 27 (VIC-MGB)

Since the wild type 93Y21 genomic DNA with two alleles of the targetsite was used as the single copy calibrator, events without any changeof the target site would be detected as one copy herein termed Wt-Homo(qPCR value >=0.7), events with one allele changed, which is no longerdetectible by the target site-specific qPCR, would be detected as halfcopy herein termed NHEJ-Hemi (qPCR value between 0.1 and 0.7), whileevents with both alleles changed would be detected as null herein termedNHEJ-Null (qPCR value=<0.1). Two soybean transformation experiments werecarried out. The Mega74 experiment was to use the RTW1211 construct toknockout only the two FAD2-1 genes. Both NHEJ-Hemi and NHEJ-Null weredetected in FAD2-1A and FAD2-1B genes (Table 3). In the second Mega82experiment, the RTW1312 was added in additional to the RTW1211 constructto knockout both the two FAD2-1 genes and the two FAD3 genes. Efficientquadra gene knockout were detected in FAD2-1A, FAD2-1B, FAD3a and FAD3bgenes (Table 4).

TABLE 3 FAD2-1 Target site mutations induced by the guide RNA/Cas9system in 93Y21. Numbers indicate number of events (numbers inparentheses are %). Total Wt-Homo NHEJ-Hemi NHEJ-Null Target site event(%) (%) (%) FAD2-1A 84 29 (35%) 49 (58%)  6 (7%) FAD2-1B 25 (30%) 23(27%) 36 (43%)

TABLE 4 FAD2-1 and FAD3 target site mutations induced by the guideRNA/Cas9 system in 93Y21. Numbers indicate number of events (numbers inparentheses are % of the total analyzed events). Total Wt-Homo NHEJ-HemiNHEJ-Null Target Site event (%) (%) (%) FAD2-1A 64  7 (11%) 15 (23%) 42(66%) FAD2-1B 11 (17%) 10 (16%) 43 (67%) FAD3a 6 (9%)  8 (13%) 50 (78%)FAD3b 2 (3%)  8 (13%) 54 (84%)

The target regions of NHEJ-Null events were amplified by regular PCRfrom the same genomic DNA samples using primers specific respectively toFAD2-1A, FAD2-1B, FAD3a and FAD3b genes (Table 5). The PCR bands werecloned into pCR2.1 vector using a TOPO-TA cloning kit (Invitrogen) andmultiple clones were sequenced to check for target site sequence changesas the results of NHEJ. Various small deletions at the Cas9 cleavagesite, 3 bp upstream of the PAM, were revealed at all four tested targetsites, with most of them resulting in frame-shift knockouts (Table 6,FIG. 1A, FIG. 1B and FIG. 2). These sequence analyses confirmed theoccurrence of NHEJ mediated by the guide RNA/Cas9 system at the specificCas9 target sites.

TABLE 5 PCR primers for the gRNA targets of the FAD2-1 and FAD3 genesTarget Site Primer1 SEQ ID NO: Primer2 SEQ ID NO: FAD2-1A WOL1007 28WOL1009 30 FAD2-1B WOL1008 29 WOL1009 30 FAD3a WOL1100 31 WOL1101 32FAD3b WOL1102 33 WOL1103 34

TABLE 6 Edited Alleles of the FAD2-1A, FAD2-1B, FAD3a and FAD3b genesVariant FAD2-1A FAD2-1B FAD3a FAD3b 1.1 11 bp del  4 bp del 1.2  7 bpdel 11 bp del 1.3  8 bp del 16 bp del 6.1  5 bp del 11 bp del 1.4  7 bpdel  1 bp insert 1.5  4 bp del  7 bp del 1.6  1 bp del  8 bp del 1.7 26bp del  8 bp del 3.1  7 bp del  5 bp del 2 bp del 2 bp del 5.3  7 bp del 7 bp del 4 bp del 2 bp del 1.5a 13 bp del  5 bp del 4 bp del 2 bp del

Example 5 Compositional Analysis of the Soybean Seeds and Identificationof Lead Variants

Screening of seed from edited events was performed using non-destructivesingle seed Near Infrared analysis (SS-NIR). The instrument used hadbeen calibrated to determine the fatty acid profiles in intact seedwhile maintaining the seeds individual identity. By so doing seeddisplaying the desired phenotype could be selected for propagation.SS-NIR calibration models were created using proprietary seed displayinga wide dynamic range in the constituents of interest, e.g., oleic andlinolenic acids, as described below. A Single Seed Near Infrared(SS-NIR) spectrometer (U.S. Pat. No. 7,274,456 B2, issued Sep. 25, 2007;U.S. Pat. No. 7,508,517B2, issued Mar. 24, 2009) was used. In the SS-NIRsystem an individual bean was introduced into the analytical cell whereit was illuminated from all points in three dimensions. The seed wastumbled with an air stream, within an approximated integrating sphereconstructed from a 16-mm-diameter quartz cup coated with 6080 whitereflectance coating (Labsphere, North Hutton, N.H.). Illumination wasprovided through 12 optical fibers, connected to four 20 Watt 8211-002light bulbs (Welch Allyn, Skaneateles Falls, N.Y.), the ends of whichwere incorporated into the cell cover. The reflected spectral regionfrom 904 to 1686 nm was collected through the apex of the cover of thesampling cell by an NIR512 spectrometer (Control Development, SouthBend, Ind.). Each seed was scanned for 6 seconds to collect spectra thatwere optimized for maximal signal to noise ratio. Spectral quality wasmonitored during each sample scan by regularly checking the Root MeanSquare (RMS) noise of the 100% lines. The 100% lines were computed bythe ratio between every two spectra of the triplicate measurement foreach sample. Under ideal, noise-free conditions, the 100% lines would bestraight horizontal lines at zero absorbance units (AU) since allreplicate spectra come from the same sample providing the same spectralfeatures. To minimize instrumental drift, system noise, seed conditionand other environmental changes, noise and off-sets were observed in theactual 100% lines. After scanning, the seed was ejected from the samplecup and transferred to an indexed sample tray. The individual identityof each seed was therefore preserved, facilitating instrumentcalibration.

Separate calibration models were generated for each constituent ofinterest using Partial Least Square (PLS) analysis coupled with anoptimized number of latent variables, spectral range and spectralpreprocessing, before being applied to online/offline compositionalanalysis of the individual seed components, such as the oleic andlinolenic acid. The optimized number of latent variables, spectral rangeand spectral preprocessing were determined by analyzing the training andmonitoring subset from the calibration data where the calibrationperformance reached an optimum level, in terms of Root Mean Square Errorof Calibration (RMSEC) and Root Mean Square Error of Cross Validation(RMSECV). For those co-constituents with distinct spectra, such as oil,a few PLS latent variables were used to capture enough information. MorePLS latent variables were needed for components with less distinctspectra. After the spectra were preprocessed for multiplicative scattercorrections, Savitsky-Golay derivatives and polynomial smoothing wereapplied in the spectral region between 904-1540 nm. The number of latentvariables was determined as the fewest number of latent variables thatresulted in an optimal calibration/cross validation accuracy asdetermined by the RMSEC (Root Mean Square Error of Calibration) andRMSECV (Root Mean Square Error of Cross Validation), respectively. Theoptimum calibration model was selected based on the R² (statisticalmeasure of how close the predicted and reference chemistry data arefitted by the regression line), RMSEC (Root Mean Square Error ofCalibration) and RMSECV (Root Mean Square Error of Cross Validation)statistics.

TABLE 7 Statistics for the oleic and linolenic Acid SS-NIR calibrations.The number of reference chemistry measurements used to develop thecalibrations for each constituent are shown in column n. The dynamicrange in composition underpinning each constituent calibration is shownin the range column. Range Constituent n (rel %-rel %) R² RMSEC RMSECVOleic acid 2725 12.8-90.1 0.99 2.80% 2.83% Linolenic acid 2725  1.1-12.70.92 0.81% 0.86% R² = regression coefficient; RMSEC = Root Mean SquareError of Calibration; RMSECV = Root Mean Square Error of CrossValidation

Thirty-six seed from each T₀ edited event were subjected to SS-NIR andthe predicted oleic and linolenic acid contents of the seed were used toidentify those carrying the desired High Oleic or High Oleic/LowLinolenic phenotype.

Sample Preparation T₂ or T₃ Homozygous Seed.

Eight soybean seeds were placed in a Spex Certiprep 13/16×2″polycarbonate vial with cap (MedPlast, Monticello, Iowa; cat #1076). A9/16″ stainless steel ball bearing was added. Grinding was performed ina Spex Certiprep 2000 Geno/Grinder at 1500 strokes/min for three 30second intervals with a 1-minute rest between each cycle. The grindingball was removed and the powder was thoroughly dispersed with a spatulaand analyzed as described below.

GC Fatty Acid Profile Determinations

Two replicate extractions were performed on each sample, as follows:

-   -   Weigh ground sample (approximately 20-30 mg; to an accuracy of        0.1 mg) into 13×100 mm tube (with Teflon® lined cap; VWR        (53283-800) and record weight.    -   Add 2 mL Heptane, vortex and place into an ultrasonic bath (VWR        Scientific Model 750D) at 60° C. for 15 min at full        sonification-power (˜360 W).    -   Centrifuge for 5 min at 1700×g at room temperature.    -   Decant the supernatant to a clean 13×100 mm glass tube.        Fatty Acid Methyl Ester Profile Determination: GC Method:    -   Transfer 1000 uL aliquot of the heptane extract into a screw top        GC vial National Scientific (C4000-186W)    -   Add 100 uL trimethylsulfonium hydroxide in methanol (JenaChem)        and cap the vial.    -   Shake the vials on an orbital shaker at room temperature for 15        minutes.    -   The fatty acid methyl esters were analyzed by directly injecting        1 uL samples (at a 5:1 split ratio) onto an Agilent 7890 gas        chromatography system fitted with a Supelco Omegawax 320        (30m×0.320 mm×0.25 um film) capillary column. Hydrogen was used        as the carrier gas (39 cm/sec average linear velocity). Inlet        and FID detector temperatures were held at 260° C. and the oven        column temperature was ramped from 180 to 240° C. at a rate of        12° C. per minute.    -   The relative percentages of the individual fatty acid methyl        esters were determined using ChemStation.        Fatty Acid Profiling in Homozygous Mega74 and Mega82 Seeds.    -   Fatty acid compositions of the homozygous Mega74 T3 seeds        (FAD2-1A/FAD2-1B knockout) grown in Johnston 2016 field test        were analyzed by the GC method described above and the results        are shown in Table 8.

TABLE 8 Fatty acid profiles (relative area %) of T3 seeds harvested fromfield grown Mega74 events, commodity (93Y21) and transgenic high oleicsoybean varieties JH89136339 (FAD2 transgene knockout) and P31T96PR(FAD2 transgenic knockout plus 2 FAD3 recessive alleles). 18:1Description Name 16:00 16:01 17:00 17:01 18:00 Total 18:02 18:03 Mega 74entry1 6.8 0.1 0.1 0.1 3.3 83.2 1.3 3.3 Mega 74 entry2 6.9 0.1 0.1 0.13.6 82.7 1.6 3.3 Mega 74 entry3 6.8 0.1 0.1 0.1 3.3 82.3 2.1 3.5 Mega 74entry4 6.9 0.1 0.1 0.1 3.5 83.0 1.4 3.2 Mega 74 entry5 6.8 0.1 0.1 0.13.5 82.6 1.5 3.4 Mega 74 entry6 6.9 0.1 0.1 0.1 3.6 82.4 1.4 3.6 Mega 74entry7 6.9 0.1 0.1 0.1 3.6 82.7 1.4 3.3 Mega 74 entry8 6.8 0.1 0.1 0.13.6 83.1 1.3 3.2 Mega 74 entry9 6.7 0.1 0.1 0.1 3.4 83.2 1.3 3.3 Mega 74entry10 6.9 0.1 0.1 0.1 3.5 82.3 2.0 3.3 Mega 74 entry11 6.9 0.1 0.1 0.13.5 83.2 1.3 3.1 Mega 74 entry12 6.8 0.1 0.1 0.1 3.5 83.1 1.3 3.3 Mega74 entry13 6.8 0.1 0.1 0.1 3.5 82.8 1.4 3.4 Mega 74 entry14 6.9 0.1 0.10.1 3.4 82.8 1.4 3.3 Mega 74 entry15 6.8 0.1 0.1 0.1 3.5 83.0 1.3 3.3Mega 74 entry16 6.7 0.1 0.1 0.1 3.5 83.4 1.2 3.1 Mega 74 entry17 6.8 0.10.1 0.1 3.6 82.8 1.4 3.4 Mega 74 entry18 6.9 0.1 0.1 0.1 3.5 83.0 1.33.2 HO parent JH89136339 6.2 0.1 0.7 1.2 3.3 80.2 2.0 4.3 PlenishP31T96PR 6.0 0.1 0.7 1.1 3.6 80.6 4.3 1.7 93Y21 93Y21 11.0 0.1 0.1 0.13.8 23.4 53.1 7.5The relative area percent is representative of a fatty acid weightpercent when expressed as a percentage of the total fatty acids byweight. The data show that the knockout of the FAD2-1A and FAD2-1B insoybean delivered a high oleic phenotype. The linolenic acid level isaround 3.1-3.6%.

Fatty acid compositions of the homozygous Mega82 T2 seeds(FAD2-1A/FAD2-1B/FAD3a/FAD3b knockout) grown in greenhouse 2016 wereanalyzed by the GC method described above and the results are shown inTable 9.

TABLE 9 Fatty acid profiles (relative area %) of T2 seeds harvested fromMega82 events in greenhouse, commodity (93Y21) and transgenic high oleicsoybean varieties JH89136339 (FAD2 transgene knockout) and P31T96PR(FAD2 transgenic knockout plus 2 FAD3 recessive alleles). 16:0 16:1 17:017:1 18:0 18:1 18:2 18:3 Description Name Area Area Area Area Area AreaArea Area Mega 82 entry1 7.6 0.1 0.1 0.1 3.9 78.1 5.8 2.1 Mega 82 entry27.6 0.1 0.1 0.1 3.5 80.4 4.8 1.3 Mega 82 entry3 7.5 0.1 0.1 0.1 3.5 80.44.9 1.3 Mega 82 entry4 7.3 0.1 0.1 0.1 3.5 80.9 4.2 1.6 Mega 82 entry57.5 0.1 0.1 0.1 3.6 80.5 4.3 1.6 Mega 82 entry6 7.3 0.1 0.1 0.1 3.7 80.74.1 1.6 Mega 82 entry7 7.6 0.1 0.1 0.1 3.8 78.6 5.9 1.7 Mega 82 entry87.3 0.1 0.1 0.1 3.7 79.8 5.1 1.7 Mega 82 entry9 7.4 0.1 0.1 0.1 4.1 77.66.3 2.1 Mega 82 entry10 7.5 0.1 0.1 0.1 3.8 80.1 4.7 1.5 Mega 82 entry117.4 0.1 0.1 0.1 4.1 78.5 5.7 1.8 Mega 82 entry12 7.6 0.1 0.1 0.1 3.778.3 5.8 1.9 Mega 82 entry13 7.7 0.1 0.1 0.1 3.5 80.4 4.4 1.6 Mega 82entry14 8.0 0.1 0.1 0.1 3.3 80.0 4.8 1.6 Mega 82 entry15 7.7 0.1 0.1 0.13.3 81.3 3.6 1.6 Mega 82 entry16 7.6 0.1 0.1 0.1 3.5 79.4 5.4 1.7 Mega82 entry17 7.6 0.1 0.1 0.1 3.6 80.2 4.5 1.6 Mega 82 entry18 7.7 0.1 0.10.1 3.5 80.9 3.8 1.6 HO parent JH89136339 6.2 0.1 0.7 1.2 3.3 80.2 2.04.3 Plenish P31T96PR 6.0 0.1 0.7 1.1 3.6 80.6 4.3 1.7 93Y21 93Y21 11.00.1 0.1 0.1 3.8 23.4 53.1 7.5

The relative area percent is representative of a fatty acid weightpercent when expressed as a percentage of the total fatty acids byweight. The data show that the knockout of the FAD2-1A, FAD2-1B, FAD3aand Fad3b genes in soybean delivered both the high oleic and linolenicphenotype.

Fatty Acid Profiling in Non-Target Tissues.

Homozygous Mega74, Mega82 events, commodity (93Y21 and 93B86) andtransgenic high oleic soybean varieties 93M02P (FAD2 transgene knockout)and P34T90PR (FAD2 transgenic knockout plus 2 FAD3 recessive alleles)were grown in short rows in the field in Johnston Iowa during the 2017growing season. Forty-two days after flowering, when the plants were atthe R5 stage, individual plants were pulled from within the test rows.Leaf, stem and washed root material were harvested, flash frozen inliquid nitrogen and transported back to the laboratory where they werestored at −80° C. until further processing.

The tissue was ground to a fine powder in a mortar and pestle in thepresence of liquid nitrogen. Twenty mg aliquots of the powders weretransferred to 13×100 mm test tubes and 2 ml of freshly prepared 5%concentrated sulfuric acid in anhydrous methanol was added to each. Thethreads on the tubes were wrapped with Teflon® pipe tape and sealed witha Teflon® lined cap. Samples were vortex mixed and heated for 1 h at 80°C. The samples were allowed to cool to room temperature and 1 ml of 1Maqueous sodium chloride was added to each tube followed by 500 ul ofheptane. The tubes were vortexed and allowed to stand. Once the phaseshad separated, 200 ul of the upper heptane phase was transferred to aGC-vial fitted with a volume reducing liner. The samples were thensubjected to fatty acid methyl ester analysis, as described above, using2 ul injections.

TABLE 10 Fatty acid profiles (relative area %) of non-target tissueharvested from field grown Mega74, Mega82 events, commodity (93Y21 and93B86) and transgenic high oleic soybean varieties 93M02P (FAD2transgene knockout) and P34T90PR (FAD2 transgenic knockout plus 2 FAD3recessive alleles). 16:0 16:1 17:0 17:1 18:0 Total 18:2 18:3 MaterialName Area Area Area Area Area 18:1 Area Area 42 DAF leaf P34T90PR 12.40.2 0.2 0.1 3.8 0.9 8.6 73.8 42 DAF leaf 93B86 11.2 0.3 0.1 0.2 3.5 2.012.5 70.3 42 DAF leaf 93Y21 11.3 0.3 0.2 0.2 3.1 2.1 11.3 71.4 42 DAFleaf 93M02P 11.4 0.2 0.1 0.1 3.6 1.9 13.2 69.5 42 DAF leaf Mega74 11.80.3 0.2 0.2 3.2 1.8 9.8 72.8 event 6.1 42 DAF leaf Mega82 11.0 0.3 0.20.2 3.2 1.5 8.6 75.1 event 3.1 42 DAF leaf Mega82 12.2 0.3 0.2 0.2 3.32.1 13.0 68.7 event 5.3 42 DAF root P34T90PR 19.2 0.7 0.7 0.2 3.6 4.651.0 20.1 42 DAF root 93B86 18.1 0.2 0.4 0.1 2.9 3.3 43.7 31.2 42 DAFroot 93Y21 16.7 0.2 0.5 0.2 3.0 2.6 42.9 34.0 42 DAF root 93M02P 17.40.4 0.5 0.1 2.9 2.4 40.6 35.7 42 DAF root Mega74 18.0 0.2 0.5 0.1 3.72.7 44.1 30.8 event 6.1 42 DAF root Mega82 17.3 0.2 0.5 0.2 3.6 2.4 53.022.8 event 3.1 42 DAF root Mega82 17.1 0.4 0.5 0.2 3.0 2.3 51.6 25.0event 5.3 42 DAF stem 93B86 16.2 0.2 0.3 0.0 3.9 3.1 33.5 42.8 42 DAFstem 93Y21 16.0 0.2 0.3 0.0 3.9 3.2 33.1 43.2 42 DAF stem 93M02P 16.50.1 0.3 0.0 3.7 2.3 31.7 45.3 42 DAF stem Mega74 16.0 0.3 0.3 0.0 3.83.4 32.9 43.3 event 6.1 42 DAF stem Mega82 16.5 0.3 0.4 0.0 5.1 1.7 33.442.7 event 3.1 42 DAF stem Mega82 15.1 0.2 0.3 0.0 4.2 2.5 36.5 41.1event 5.3

The relative area percent is representative of a fatty acid weightpercent when expressed as a percentage of the total fatty acids byweight. The data show that the off-target (non-seed) tissues in theMega74 and Mega82 events had very low levels of oleic acid, which wereindistinguishable from those in the comparable tissues harvested fromthe control tissues varieties. Subtle differences in the linoleic (18:2)and linolenic (18:3) acid contents of the root tissues were apparent.The Mega82 materials, which carry FAD3a and FAD3b knockouts, had higherlinoleic and lower linolenic acid contents than Mega74, the commoditysoy varieties (93B86 and 93Y21) and the transgenic FAD2 knockout variety93M02P. High oleic variety P34T90PR, which carries two recessive FAD3alleles in addition to a transgenic knockout of the FAD2 alleles, had asimilar fatty acid profile the two Mega82 events.

Example 6 Agronomic Evaluation of CRISPR-Edited High-Oleic Low-LinolenicVariants

Seeds of the three soybean variants 3.1, 5.3 and 1.5a (each containingthe FAD2-1A, FAD2-1B, FAD3a and FAD3b mutation edits shown in FIG. 2which were generated simultaneously in each variant using two guideRNAs) were grown in the field at three varied locations in the UnitedStates alongside a 93Y21 elite soybean control to assess agronomicperformance of the variants. Emergence, plant height, maturity and yieldwere measured and averaged across the locations. No measurabledifference could be detected between the variants and the control foremergence, plant height, maturity and yield. Protein and oil was alsomeasured in seeds harvested from the plants grown in the three variedlocations. Seeds of the three variants 3.1, 5.3 and 1.5a were found tohave comparable oil and protein content on a seed dry weight basis asthe control 93Y21 seeds and control seeds having a FAD2 transgenicknockout plus 2 FAD3 recessive alleles.

Example 7 Detection of Site-Specific Mutations with NHEJ Mediated by theGuide RNA/Cas9 System in Stably Transformed Soybean

Genomic DNA was extracted from somatic embryo samples and analyzed byquantitative PCR using a 7500 real time PCR system (Applied Biosystems,Foster City, Calif.) with target site-specific primers and a FAM-labeledfluorescence probe to check copy number changes of the target sites. TheqPCR analysis was done in duplex reactions with a syringolide inducedprotein (SIP) gene as the endogenous control and a wild type 93Y21genomic DNA sample that contains one copy of the target site with 2alleles, as the single copy calibrator. The endogenous control probeSIP-T was labeled with VIC(2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein) and thegene-specific probes FAD2-T1, FAD2-T2 and FAD3-T2 were labeled with FAM(Fluorescein) (Table 11) for the simultaneous detection of bothfluorescent probes (Applied Biosystems). PCR reaction data were capturedand analyzed using the sequence detection software provided with the7500 real time PCR system and the gene copy numbers were calculatedusing the relative quantification methodology (Applied Biosystems).

TABLE 11 Primers/probes used in qPCR analyses of transgenic soybeanevents. Target Primer/Probe SEQ ID Site Name Sequences NOs: FAD2-1AFAD2-F1 TCGTGTGGCCAAAGTGGAA 11 FAD2-R1 TTTGTGTTTGGAACCCTTGAGA 12 FAD2-T1TTCAAGGGAAGAAGCC 13 (FAM-MGB) FAD2-1B FAD2-F2 CCGTGTGGCCAAAGTTGAA 14FAD2-R1 TTTGTGTTTGGAACCCTTGAGA 12 FAD2-T2 TTCAGCAGAAGAAGCC 15 (FAM-MGB)FAD3a FAD3-F1 TAATGGATACCAAAAGGAAGC 16 FAD3-R2 CAAGCACATCCCTGAGAACATAA17 C FAD3-T2 AATCCATGGAGATCCCT 18 (FAM-MGB) FAD3b FAD3-F2ATACCAACAAAAGGGTTCTTC 19 FAD3-R2 CAAGCACATCCCTGAGAACATAA 17 C FAD3-T2AATCCATGGAGATCCCT 18 (FAM-MGB) SIP SIP-130F TTCAAGTTGGGCTTTTTCAGAAG 80SIP-198R TCTCCTTGGTGCTCTCATCACA 81 SIP-170T CTGCAGCAGAACCAA 82 (VIC-MGB)

Since the wild type 93Y21 genomic DNA with two alleles of the targetsite was used as the single copy calibrator, events without any changeof the target site would be detected as one copy herein termed Wt-Homo(qPCR value >=0.7), events with one allele changed, which is no longerdetectible by the target site-specific qPCR, would be detected as halfcopy herein termed NHEJ-Hemi (qPCR value between 0.1 and 0.7), whileevents with both alleles changed would be detected as null herein termedNHEJ-Null (qPCR value=<0.1).

In total, four soybean transformation experiments were carried out. InRV019927 and RV019929, the gRNA cassettes were near the right border andplaced upstream of the Cas9 expression cassettes in the binary vectors.In contrast, in RV019928 and RV019930, the gRNA expression cassetteswere near the left border and placed downstream of the Cas9 expressioncassettes in the binary vectors. As shown in Table 12, Table 13, for thetwo experiments to knockout only the two FAD2-1 genes, the gRNA near theright border and placed upstream of Cas9 configuration design providedmuch higher gene knockout efficiency as compared to the design with thegRNA near the left border and placed downstream of Cas9. For example,the bi-allelic (NHEJ-Null) knockout efficiency reached 63% for theFAD2-1A gene and 56% for the FAD2-1B gene (Table 12) with the RV019927vector. The bi-allelic (NHEJ-Null) knockout efficiency was only 7% forthe FAD2-1 gene and 10% for the FAD2-1B gene (Table 13) with theRV019928 vector. The WT population only made up about 2-3% in theexperiment with the RV019927 vector, as compared to 37-44% in theexperiment with the RV019928 vector.

In the third and fourth experiments, either the RV019929 or RV019930binary vectors was used to knockout the two FAD2-1 genes and the twoFAD3 genes. As shown in Table 12, Table 13, these two experiments alsodemonstrated that RV019929 vector design, with the gRNA expressioncassette near the right border and upstream of the Cas9 expressioncassette, provided much higher quadra gene knockout efficiency ascompared to the RV019930 binary vector in the FAD2-1A, FAD2-1B, FAD3aand FAD3b genes. These unexpected results demonstrated the differentgRNA/Cas9 expression cassette configurations in the binary vectors haddramatic effects on the target gene editing efficiency. Vectors withgRNA cassettes near the right border and placed upstream of the Cas9expression cassettes increased the efficiency of editing the targetsite.

TABLE 12 FAD2-1 Target Site Mutations Induced by the Guide RNA/Cas9system in 93Y21 with RV019927. Numbers indicate no. of events (numbersin parentheses are %). Wt-Homo NHEJ-Hemi NHEJ-Null Target site Totalevents (%) (%) (%) FAD2-1A 106 2 (2%) 37 (35%) 67 (63%) FAD2-1B 3 (3%)44 (42%) 59 (56%)

TABLE 13 FAD2-1 Target Site Mutations Induced by the Guide RNA/Cas9system in 93Y21 with RV019928. Numbers indicate no. of events (numbersin parentheses are %). Wt-Homo NHEJ-Hemi NHEJ-Null Target site Totalevent (%) (%) (%) FAD2-1A 41 15 (37%) 23 (56%) 3 (7%)  FAD2-1B 18 (44%)19 (46%) 4 (10%)

TABLE 14 FAD2-1 and FAD3 Target Site Mutations Induced by the GuideRNA/Cas9 system in 93Y21 with RV019929. Numbers indicate no. of events(numbers in parentheses are % of the total analyzed events). TotalWt-Homo NHEJ-Hemi NHEJ-Null Target Site event (%) (%) (%) FAD2-1A 27 0(0%) 11 (41%) 16 (59%) FAD2-1B 1 (4%) 12 (44%) 14 (52%) FAD3a 1 (4%)  6(22%) 20 (74%) FAD3b 1 (4%)  4 (15%) 22 (81%)

TABLE 15 FAD2-1 and FAD3 Target Site Mutations Induced by the GuideRNA/Cas9 system in 93Y21 with RV019930. Numbers indicate no. of events(numbers in parentheses are % of the total analyzed events). TotalWt-Homo NHEJ-Hemi NHEJ-Null Target Site event (%) (%) (%) FAD2-1A 42 20(48%) 17 (40%) 5 (12%) FAD2-1B  7 (17%) 32 (76%) 3 (7%)  FAD3a  7 (17%)32 (76%) 3 (7%)  FAD3b 12 (29%) 22 (52%) 8 (19%)

The target regions of NHEJ-Null events were amplified by regular PCRfrom the same genomic DNA samples using primers specific respectively toFAD2-1A, FAD2-1B, FAD3a and FAD3b genes (Table 8).

TABLE 8 PCR primers for the gRNA targets of the FAD2-1 and FAD3 genesTarget Site Primer1 SEQ ID NO: Primer2 SEQ ID NO: FAD2-1A WOL1007 23WOL1009 25 FAD2-1B WOL1008 24 WOL1009 25 FAD3a WOL1100 26 WOL1101 27FAD3b WOL1102 28 WOL1103 29

The PCR bands were cloned into pCR2.1 vector using a TOPO-TA cloning kit(Invitrogen) and multiple clones were sequenced to check for target sitesequence changes as the results of NHEJ. Various small deletions nearthe Cas9 cleavage site, 3 bp upstream of the PAM, were revealed at allfour tested target sites, with most of them resulting in frame-shiftknockouts (FIG. 3A-FIG. 3B, FIG. 4A-FIG. 4B, FIG. 5A-FIG. 5B). Thesesequence analyses confirmed the occurrence of NHEJ mediated by the guideRNA/Cas9 system at the specific Cas9 target sites.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding. Asis readily apparent to one skilled in the art, the foregoing disclosuresare only some of the methods and compositions that illustrate theembodiments of the foregoing invention. It will be apparent to those ofordinary skill in the art that variations, changes, modifications, andalterations may be applied to the compositions and/or methods describedherein without departing from the true spirit, concept, and scope of theinvention.

All publications, patents, and patent applications mentioned in thespecification are incorporated by reference herein in their entiretiesfor the purpose cited to the same extent as if each was specifically andindividually indicated to be incorporated by reference herein.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth. Unless expressly stated to the contrary, “or” is used as aninclusive term. For example, a condition A or B is satisfied by any oneof the following: A is true (or present) and B is false (or notpresent), A is false (or not present) and B is true (or present), andboth A and B are true (or present).

What is claimed is:
 1. A method of altering the fatty add profile in theseed of a soybean plant, the method comprising introducing four or morenucleotide modifications through four or more targeted DNA breaks atfour or more genomic loci of a plant at the same time, wherein (a) thegenomic loci comprise a first polynucleotide encoding a FAD2-1Apolypeptide, a second polynucleotide encoding a FAD3a polypeptide, athird polynucleotide encoding a FAD2-1 B polypeptide, and a fourthpolynucleotide encoding a FAD3b polypeptide, and (b) wherein the oleicacid content is increased to at least 75% by weight of the total fattyacids and the linolenic acid content is decreased to less than 3% byweight of the total fatty acids in the seed compared to the seed of acontrol plant not comprising the four or more introduced nucleotidemodifications.
 2. The method of claim 1, wherein the nucleotidemodifications are introduced by targeted DNA breaks using no more thantwo guide RNAs.
 3. The method of claim 1, wherein the soybean plant hasa similar yield as the control plant.
 4. The method of claim 1, whereinthe modifications target the genomic locus at a target site such thatmore than one genetic modifications at different sites are presentwithin (a) the same coding region; (b) non-coding region; (c) regulatorysequence; or (d) untranslated region of an endogenous polynucleotideencoding a polypeptide that is involved in fatty acid metabolism.
 5. Themethod of claim 1, wherein the target site comprises SEQ ID NO: 6 or SEQID NO:
 7. 6. The method of claim 1, wherein the first polynucleotidecomprises at least one of SEQ ID NOS: 35-43 wherein the secondpolynucleotide comprises at least one of SEQ ID NOS: 59-61, wherein thethird polynucleotide comprises at least one of SEQ ID NOS: 44-58, andwherein the fourth polynucleotide comprises at least one of SEQ ID NOS:62-64.
 7. The method of claim 1, wherein the first polynucleotideencodes a FAD2-1A polypeptide having at least 90% identity to SEQ ID NO:70, the second polynucleotide encodes a FAD3a polypeptide haying atleast 90% identity to SEQ ID NO: 74, and wherein the genomic locicomprise a third polynucleotide encoding a FAD2-1 B polypeptide havingat least 90% identity to SEQ ID NO: 72 and a fourth polynucleotideencoding a FAD3b polypeptide having at least 90% identity to SEQ ID NO:76.
 8. The method of claim 7, wherein the first polynucleotide comprisesSEQ ID NO: 54, the second polynucleotide comprises SEQ ID NO: 60, thethird polynucleotide comprises SEQ ID NO: 57, and the fourthpolynucleotide comprises SEQ ID NO:
 63. 9. The method of claim 7,wherein the first polynucleotide comprises SEQ ID NO: 55, the secondpolynucleotide comprises SEQ ID NO: 61, the third polynucleotidecomprises SEQ ID NO: 58, and the fourth polynucleotide comprises SEQ IDNO:
 64. 10. The method of claim 1, wherein the genomic loci comprise anedit in a polynucleotide that encodes a FAD3 polypeptide comprising anamino acid sequence that is at least 90% identical to a sequenceselected from the group consisting of SEQ ID NOS: 74 or 76 such that theedit results in one or more of the following: (a) reduced expression ofa polynucleotide encoding the FAD3 polypeptide; (b) reduced activity ofthe FAD3 polypeptide; (c) generation of one or more alternative splicedtranscripts of a polynucleotide encoding the FAD3 polypeptide; (d)deletion of one or more active sites of the FAD3 polypeptide; (e)frameshift mutation in one or more exons of a polynucleotide encodingthe FAD3 polypeptide; (f) deletion of a substantial portion of thepolynucleotide encoding the FAD3 polypeptide or deletion of thepolynucleotide encoding the full-length FAD3 polypeptide; (g) repressionof an enhancer motif present within a regulatory region encoding theFAD3 polypeptide; and (h) modification of one or more nucleotides ordeletion of a regulatory element operably linked to the expression ofthe polynucleotide encoding the FAD3 polypeptide, wherein the regulatoryelement is present within a promoter, intron, 3′UTR, terminator or acombination thereof.
 11. The method of claim 1, wherein the genomic locicomprise an edit in a polynucleotide that encodes a FAD2-1 polypeptidecomprising an amino acid sequence that is at least 90% identical to asequence selected from the group consisting of SEQ ID NOS: 70 or 72 suchthat the edit results in one or more of the following: (a) reducedexpression of a polynucleotide encoding the FAD2-1 polypeptide; (b)reduced activity of the FAD2-1 polypeptide; (c) generation of one ormore alternative spliced transcripts of a polynucleotide encoding theFAD2-1 polypeptide; (d) deletion of one or more domains of the Br2polypeptide; (e) frameshift mutation in one or more exons of apolynucleotide encoding the FAD2-1 polypeptide; (f) deletion of asubstantial portion of the polynucleotide encoding the FAD2-1polypeptide or deletion of the polynucleotide encoding the FAD2-1polypeptide; (g) repression of an enhancer motif present within aregulatory region encoding the FAD2-1 polypeptide; (h) modification ofone or more nucleotides or deletion of a regulatory element operablylinked to the expression of the polynucleotide encoding the FAD2-1polypeptide, wherein the regulatory element is present within apromoter, intron, 3′UTR, terminator or a combination thereof.
 12. Themethod of claim 1, wherein the targeted DNA break is not effected usinga TALEN (transcription activator-like effector nuclease).